In 1940, Avery once again focused on the problem of bacterial transformation. After Colin MacLeod left to join the faculty at New York University later that year, Maclyn McCarty joined the laboratory in 1941 and aided Avery in this research. Avery and McCarty focused first on purifying the transforming substance. Using refined versions of Colin M. MacLeod's preparation techniques, Avery and McCarty isolated biologically active "transforming principle" from samples of pneumococci. After jump-starting the research, Avery was increasingly preoccupied with the step-by-step purification of the transforming agent and its identification. Initially, transformation had been a tentative and delicate phenomenon that was difficult to consistently recreate. Avery later told Rollin Hotchkiss, "Many are the times we were ready to throw the whole thing out the window!" Eventually, Avery and McCarty were able to take a culture of pneumococci of an R form that had been attenuated from an S of Type II over the course of thirty generations, and add to it to the highly purified deoxyribonucleic acid (DNA) extracted from an S of Type III. This process resulted, by the next generation, in large and fully developed colonies of S Type III, which remained stable through several generations. After achieving reliable and long-lasting transformation, Avery turned to prove that it was caused by DNA alone, despite the prevailing conviction of most geneticists, and even his own earlier belief, that DNA was a simple molecule and that genes must be composed of protein, a seemingly more complex substance.

As Avery and McCarty turned their attention to the chemical analysis of transformation, they found that proteases (enzymes that deactivate proteins) and lipases (enzymes that destroy lipids) did not inactivate the transforming principle, and thus concluded that the substance was essentially protein- and lipid-free. However, it was not a carbohydrate like the polysaccharide capsular material, as carbohydrates are not precipitated by alcohol, as was the "transforming principle." They soon determined that the substance was rich in nucleic acids, but ribonuclease, an enzyme that destroys ribonucleic acid (RNA), did not inactivate the substance either. Further, the transforming substance had a high molecular weight, as did DNA, and gave a strong reaction to the Dische diphenylamine test, which detects for the presence of DNA. Avery and McCarty concluded that the transforming substance, which produced permanent, heritable change in an organism, was DNA.

Although the paper describing their extraordinary findings was not submitted until December of 1943, and published in the spring of 1944, Avery, McCarty, and MacLeod had accumulated all the basic experimental information and presented it to the Rockefeller Institute's Board of Scientific Directors by early April 1943. Avery anticipated significant skepticism of their claim of genetic specificity for DNA, and was weary of a repeat of the turmoil caused by his work with Alphonse Dochez on antiblastic immunity several decades earlier. Therefore he submitted the manuscript to several months of review and scrutiny by associates at the Hospital. What is more, despite Avery's confidence in their purification technique, he included in the final paper several cautionary statements that acknowledged the possibility that "the biological activity of the substance described is not an inherent property of the nucleic acid, but is due to minute amounts of some other substance absorbed to it or so intimately associated with it as to escape detection."

Avery's brother Roy was one of the first persons outside of the Hospital to be informed of their findings. In May 1943, Avery wrote his brother to inform him that he would need to delay his retirement and provide an update on the exciting developments of the previous two years of research. Avery noted that he and McCarty seemed to have identified the transforming substance: "In short, this substance is highly reactive and on elementary analysis conforms very closely to the theoretical values of pure desoxyribose nucleic acid . . . Who could have guessed it?" Noticeably excited by the finding's larger implications, Avery later told his brother that, "It touches the biochemistry of the thymus type of nucleic acids which are known to constitute the major part of chromosomes but have been thought to be alike regardless of origin and species. It touches genetics, enzyme chemistry, cell metabolism and carbohydrate synthesis." In his letter to his brother, it is evident that Avery clearly recognized the scientific importance his research with MacLeod and McCarty.

In 1944, Avery, MacLeod, and McCarty published their discovery that the transforming principle was DNA in "Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types," in the Journal of Experimental Medicine. Although their findings were clearly revolutionary, their conclusions in this paper were cautious, and they presented several interpretations of their results. The phenomenon of transformation, Avery wrote, "has been interpreted from a genetic point of view. The inducing substance has been likened to a gene, and the capsular antigen which is produced in response to it has been regarded as a gene product." Yet they also gave another interpretation, that there might be an "analogy between the activity of the transforming agent and that of a virus." However, they concluded that, "the transformation described represents a change that is chemically induced and specifically directed by a known chemical compound. If the results of the present study on the chemical nature of the transforming principle are confirmed, then nucleic acids must be regarded as possessing biological specificity." Their findings were accepted almost immediately by some, but for several years they would be the source of considerable debate among genetic researchers.

The entry of the United States into World War II and the exciting developments on the purification of the transforming substance caused Avery to delay his retirement to Tennessee past his 65th birthday in 1943. In 1944, Avery wrote his brother to inform him that, "If the war wasn't on I tell you frankly I would liquidate my affairs here [and] start for Nashville this fall." With the war came a rapid increase in the size of the U.S. military and renewed fears of a repeat of the influenza epidemic that struck soldiers during the First World War. In May of 1942, Avery joined MacLeod on the Army's Board for the Investigation and Control of Influenza and Other Epidemic Diseases as a civilian "consultant," and later sat on the Army Epidemiology Board when it was formed in 1944. He eventually retired in 1948 and moved to Nashville to be closer to his brother's family.