Nirenberg, who was born in New York City, graduated from the University of Florida in 1948 and gained his PhD in biochemistry from the University of Michigan in 1957. He then joined the National Institutes of Health in Bethesda, Maryland, where he began the work that culminated in the cracking of the genetic code.

When Nirenberg began this research it had already been surmised that different combinations of three nucleotide bases (triplets) each coded for a specific amino acid and that through the operation of this ‘genetic code’ amino acids are aligned in the right order to make proteins. The big question was; which of the 64 possible combinations of triplets codes for each of the 20 amino acids? Severo Ochoa's discovery of the technique to synthesize RNA artificially enabled Nirenberg to make an RNA molecule consisting entirely of uracil nucleotides. Thus the only triplet possible would be a uracil triplet (UUU of the code). Nirenberg found that the protein made by this RNA molecule consisted entirely of the amino acid phenylalanine, indicating that UUU must code for phenylalanine. With this first important step completed others were quick to unravel the rest of the code.

Nirenberg received the 1968 Nobel Prize for physiology or medicine for this work, sharing the award with Har Gobind Khorana and Robert Holley.