Daniel Nathans had an early interest in, and aptitude for, science and math, and majored in chemistry at the University of Delaware. His father, who very much wanted a doctor in the family, strongly encouraged his youngest child to channel this interest into medicine. Nathans later recalled that he chose the Washington University School of Medicine (WUSM) at random, not realizing that its strong program in basic sciences research--modeled after the Johns Hopkins School of Medicine--would change his plan to become a general practitioner in Wilmington. WUSM's faculty included biochemists Carl and Gerty Cori, who won the 1947 Nobel Prize for their work on glucose metabolism, biochemist Arthur Kornberg, who would soon receive a Nobel for synthesizing viral DNA, eminent microbiologist J. J. Bronfenbrenner, and pharmacologist Oliver Lowry, who developed innovative techniques for measuring minute quantities of proteins and other biological substances.

Following his first year of medical school in 1951, Nathans took a summer job in Lowry's lab. He later noted that his time with "Ollie" "really opened up for me a picture of what doing research was like, how enjoyable it was, the kinds of questions that could be asked and sometimes answered . . ." Working side by side with Lowry, Nathans determined the distribution of ascorbic acid (vitamin C) in the adrenal glands of rats, using his mentor's micro-chemical methods. He soon decided that academic medicine, where he could do lab research and teach in addition to treating patients, would suit him better than a full-time medical practice. The inspiring support and examples provided by other WUSM mentors, such as W. Barry Wood, also pulled him in that direction.

After receiving his MD in 1954, Nathans served his internship at Columbia Presbyterian Hospital in New York, under Robert Loeb, who was well-known for his clinical and laboratory studies of Addison's disease. There he was able to apply his excellent scientific and medical grounding to real problems in medicine. Nathans enjoyed the challenges of diagnosing and treating the wide variety of patients and ailments under his care, and regarded his internship year as one of the most valuable of his life. His internship experience seemed to confirm that academic medicine would be a fulfilling career path for him.

Wanting to get more research experience before serving his medical residency, Nathans became a clinical associate at the National Institutes of Health (NIH) in 1955. At the National Cancer Institute, he cared for patients enrolled in a clinical study of chemotherapy for solid tumors and for leukemia. (He later recalled that this was sometimes daunting and depressing, because the chemotherapy was primitive and not very effective, but he admired the researchers' courage and persistence.) During his second year there he worked mainly on a research project, trying to discover whether a protein often found in multiple myeloma (a cancer affecting the plasma cells, which produce antibodies, important components of the immune system) was actually produced by the tumor itself or was a response of other tissues to the presence of the tumor.

Nathans' NIH colleagues Michael Potter, John Fahey, and H. I. Pilgrim had noted that an induced mouse plasma-cell tumor, known as X5563, was associated with high concentrations of a certain immunoglobulin (antibody) protein in the blood plasma as well as other pathological changes similar to multiple myeloma in man, and this suggested an experimental approach to the question. Nathans administered a radioactively labeled amino acid (lysine) to mice with induced X5563 tumors and followed its incorporation into the tumor cells and the plasma by purifying the myeloma protein from each. By measuring the specific activity of the radioactive lysine over time in the samples, he was able to show that the protein was produced by the tumor cells rather than the non-tumor cells of the mouse.

Knowledge of protein chemistry had expanded during the 1930s and 1940s; chemists had found over twenty common amino acids (the building blocks of protein) and learned that most biochemical processes depended on proteins, especially in the form of enzymes. New techniques such as chromatography and electrophoresis (which separate molecules into characteristic patterns on a paper or gel medium) made it possible to better analyze protein structures. However, the exact mechanism by which cells manufacture proteins was still being unraveled in the late 1950s. The structure of DNA, established by Francis Crick and James Watson, suggested a code, and Paul Zamecnik and Mahlon Hoagland had shown that protein synthesis takes place in the ribosomes, but the role of various RNA molecules (later called "messenger" and "transfer" RNA) wasn't clear. Nathans became fascinated with the problems of protein biosynthesis during his NIH fellowship, and hoped to continue this work even as he began a two-year medical residency at Columbia Presbyterian Hospital in 1957. In his second year he was able to devote part of his time to research. By 1959, he had decided to pursue the research full time, and became a research associate at the Rockefeller Institute (later Rockefeller University), working in Fritz Lipmann's lab.

Lipmann, who won the Nobel Prize in 1953 for demonstrating the key role of adenosine triphosphate (ATP) in biochemical reactions, was intensely interested in the chemical energy exchanges of protein synthesis. He suggested that Nathans continue working with the myeloma tumor extracts, and see if he could get the extracts to make the myeloma protein. By this time Paul Zamecnik's lab had synthesized protein in cell-free systems, transfer RNA (tRNA) had been discovered, and Paul Berg had shown how amino acids are activated and bound to tRNA for incorporation into proteins. Nathans wasn't able to make the myeloma protein, but he did find and purify a protein factor that stimulated the transfer of activated amino acids from tRNA to new protein. He shifted his investigation to E. coli bacteria, (which were easier to work with than animal tumor cells, and by that time, better understood) and again found soluble protein factors that were required for activated amino acids to be added to growing peptide chains in the ribosomes. While in Lipmann's lab, he also collaborated with Amos Neidle at Columbia University to clarify the way the antibiotic puromycin inhibits protein synthesis.

Another collaboration, with Norton Zinder, James Schwartz, and Gur Notani at Rockefeller helped resolve a key question in molecular biology: how do the genetic instructions contained in DNA (located in the cell nucleus) get translated into protein synthesis by the cell's machinery (outside the nucleus)? Marshall Nirenberg and J. H. Matthei had found that RNA (RNA fractions from cells, synthetic RNA, or viral RNA) could stimulate amino acid incorporation into certain as-yet-unidentified products in E. coli extracts. They presumed these products were proteins whose structure was uniquely determined by the RNA, but had not yet verified this. Zinder had recently found an E. coli virus, coliphage f2 that had RNA rather than DNA as its genetic material. Viruses consist mostly of a protein coat or capsule and a small segment of RNA or DNA, thus much of what the genetic material codes for is the coat protein. Zinder and Nathans reasoned that if RNA from such a virus could stimulate protein synthesis, the protein produced could be identified as a component of the virus. Their study showed that the protein produced was indeed viral coat protein, and that the RNA had carried the genetic instructions for it. The experiment provided further support for the rapidly emerging idea among researchers (including Sydney Brenner, Francis Crick, Walter Gilbert, Jacques Monod, and James Watson) that a "messenger" RNA was the intermediary between DNA and protein synthesis. Nathans found this work "intoxicating" and, after three years at the Rockefeller Institute, gave up his plan to become an academic physician, and looked for a position in a basic science department at a medical school.