Bovine heart MF1-ATPase was labeled with limiting amounts of [14C]NBD-C1[( 14C]4-chloro-7-nitro-2,1,3-benzoxadiazole) and the resulting radioactive label on the essential Tyr was stabilized by reduction with zinc in the presence of multidentate ligand EDTA and redox mediator 4,4'-dipyridyl. Subsequent treatment of the labeled protein with cyanogen bromide and separation of the reaction mixture by ion-exchange chromatography yielded essentially only one radioactive polypeptide. Further cleavage of this polypeptide with TPCK-trypsin, lactonization of the terminal homoserine residue and reaction with derivatized polystyrene resin gave a shorter peptide attached to the solid support which contained all the radioactivity. Edman degradation showed that the amino acid sequence of this peptide was Glu . Gly . Asn . Asp . Leu . Tyr . His . Glu . Met, which corresponds to residues 192-200 in the beta subunit of bovine heart MF1-ATPase as determined by Runswick and Walker (1983). Since this specifically labeled Tyr-197 is separated by only one amino acid residue from the essential Glu-199 which was labeled specifically with dicyclohexylcarbodiimide by Yoshida et al. (1982) it seems most likely that both Tyr-197 and Glu-199 play direct roles in the catalytic hydrolysis and synthesis of ATP.