Several studies have been performed on the structure of muscle pyruvate kinase. X-ray diffraction has provided a three-dimensional picture of the active site, and chemical modification studies have revealed essential amino acid residues for substrate binding or catalysis. We have shown that 8-azido-ADP (N3 ADP) behaves as a photoaffinity label for the enzyme. This reagent upon irradiation produces inactivation of the enzyme, and the activity loss is protected by nucleotides. The partially modified enzyme shows the same Km for ADP as the native one suggesting an "all or none" inactivation effect. The incorporation of 1 mole of 14C-N3 ADP per subunit correlates with complete inactivation. A radioactive peptide was isolated from the enzyme labeled with 14C-N3 ADP. The partial sequence of this peptide showed that it corresponds to the same peptide isolated from rabbit muscle pyruvate kinase labeled with dialdehyde-ADP and with trinitrobenzenesulfonate. This peptide is identical to a region in the cat and chicken muscle enzymes, and also a high degree of homology is found in a region of the rat liver and yeast enzymes. These studies show that N3 ADP binds to the same site as dialdehyde-ADP in rabbit muscle pyruvate kinase, and this site seems to be the nucleotide binding site.