The chemical mechanism by which the F1 moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (Vi), a phosphate analog which has the potential of "locking" an ATPase, in its transition state by forming a MgADP.Vi complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the gamma-phosphate of ATP. Upon incubating purified rat liver F1 with MgADP and Vi for 2 h to promote formation of a MgADP.Vi-F1 complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F1 (i.e. alpha, beta, gamma, delta, and epsilon), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the beta subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F1-ATPases and the third residue within the nucleotide binding consensus GX4GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP. Vi-F1 complex revealed about 1.0 mol/mol F1, while quantification of the peptide cleavage products revealed that no more than one beta subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [3H] from NaB[3H]4 incorporates into MgADP.Vi-F1 complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single beta subunit within F1-ATPases and implicate alanine 158 as residing very near the gamma-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX4GK(T/S) sequence of many other nucleotide-binding proteins.