Despite their initial success, Nirenberg and Matthaei's newfound celebrity produced some unintended consequences. While the poly-U experiment was proof that they had in effect "cracked" the genetic code, many scientists--especially the 1959 Nobel Laureate Severo Ochoa--were eager to take it to the next level. Once it was understood that UUU was the RNA "code word" for phenylalanine, scientists set out to discover the unique code words for the twenty major amino acids. With this knowledge, scientists theorized, one would know not only how RNA translates messages from DNA to build proteins, but one would be able to read the entire genetic code of living organisms. As Jerard Hurwitz and J. J. Furth asserted in Scientific American in 1962, understanding the genetic code would explain how "the dream of the gene [becomes] the reality of the protein." Many believed, as the New York Times suggested in 1961, that "the chemical code of inheritance, which determines the form and function of every living thing and thereby provides the basis for genetics, will be cracked before the year is out."

Suddenly, the young NIH scientists found themselves in a research race with some of the world's most famous (and well-funded) molecular geneticists and biochemists, notably Ochoa at New York University School of Medicine. Over the course of the next five years, Nirenberg worked steadily with a team of about twenty postdoctoral researchers and technicians (some pitching in from other NIH labs.) Using the three-letter poly-U experiment as a kind of paradigm, the Nirenberg team extended its experiments with synthetic RNA even further, finding that poly-C (three cytosines) stimulated the production of proline. They could not, however, get poly-G (three guanines) or poly-A (three adenines) to produce amino acids. They discovered that by replacing one or two units of a triplet with other nucleotides, they could direct the production of other amino acids. They found, for example, that a synthetic RNA composed of one unit of guanine (G) added to two units of uracil (UU) directed that valine be added to a developing amino acid chain. In Nirenberg's shorthand method, the code word for valine was GUU. Concurrently, Ochoa’s group (including Robert S. Gardner, Albert J. Wahba, Carlos Basilio, Robert S. Miller, Joseph F. Speyer, and Peter Lengyel) discovered that that poly-A did code for an amino acid (lysine) if a different method was used, and found many synthetic RNA nucleotide combinations that coded for other amino acids.

In the late 1950s, the biochemist Sydney Brenner coined the term "codon" to describe the fundamental units engaged in protein synthesis, even though the units had yet to be fully determined. Francis Crick popularized the term in 1959. After 1962, Nirenberg began to use "codon" to characterize the three-letter RNA code words. With one of each of the four nucleotides occupying a place in a three-letter codon arrangement, Nirenberg quickly deduced that there were 64 possible combinations (4 x 4 x 4) of three-letter codons. Viewers can see some of Nirenberg's original charts that show the process by which he kept track of the various combinations of nucleotides in the RNA codons in the Documents section. In 1964 and 1965, Nirenberg's postdoctoral researcher, Philip Leder, developed a sophisticated filtration machine that helped the team determine the order of the nucleotides in the codons. This development speeded up the process of assigning code words to amino acids. By 1966, Nirenberg announced that he had deciphered the sixty-four RNA codons for all twenty amino acids. This remarkable personal and scientific accomplishment held great significance, not only for Nirenberg but also for the history of modern science. At a 1966 conference, Geoffrey Zubay, a professor of biology at Columbia University, remarked that "Francis [Crick] ... predicted the entire code would be solved in [1961]--and it has taken a few years longer than that and the pace, in spite of [Crick's] prediction, has been miraculously fast. I think it fair to say that Marshall Nirenberg has carried the ball all the way."

In a 1967 talk, Nirenberg characterized messenger RNA as a "robot" whose purpose was to obey the commands of DNA and carry out vital genetic instructions. "Man," Nirenberg observed, "now understands the language of the civilization, has written quite elementary messages in the form that robots understand, and via such texts has communicated directly with the robot. The robots read and faithfully carry out the instructions." For others, however, the idea of a code that controls our genetic make-up depended on less futuristic metaphors. As the 1958 Nobel Laureate George Beadle and wife Muriel Beadle wrote in 1966, "the deciphering of the DNA code has revealed our possession of a language much older than hieroglyphics, a language as old as life itself, a language that is the most living language of all--even if its letters are invisible and its words are buried deep in the cells of our bodies."

In October 1968, Nirenberg received the news that he had won the Nobel Prize in Medicine or Physiology, an honor that he shared with Robert W. Holley and Har Gobind Khorana for their collective efforts in deciphering different aspects of the genetic code. Viewers can see a photograph of Nirenberg celebrating in the laboratory. For scientists and non-scientists alike, this achievement fulfilled the predictions made just seven years earlier, when two unknown NIH scientists had first announced the poly-U experiment.