The inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoylinosine (FSBI) proceeds with pseudo-first order kinetics. The rate of inactivation increased from pH 7 to 9 revealing a pKa of about 8.2. When a tryptic digest of the enzyme which had been inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine ([3H]FSBI) was submitted to reversed phase high pressure liquid chromatography, a single major peak of radioactivity, T1, was resolved. Amino acid sequence analysis of purified peptide fragments derived from T1 showed that the modification of beta-Tyr-345 is responsible for inactivation of the enzyme. Complete inactivation of the enzyme by [3H]FSBI is estimated to proceed with modification of 0.8 mol of beta-Tyr-345/mol of enzyme. Another notable observation is that inosine triphosphatase (ITPase) activity catalyzed by F1 from bovine heart mitochondria is much more sensitive to inactivation by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) than is ATPase activity. Whereas complete inactivation of ATPase activity by FSBA has been shown to proceed with the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the beta subunit (Bullough, D. A., and Allison, W. S. (1986) J. Biol. Chem. 261, 5722-5730), it is shown here that complete inactivation of ITPase activity by FSBA is accompanied by modification of these residues in only one copy of the beta subunit. Inactivation of both the ATPase and ITPase activities of the enzyme by FSBI proceeds with modification of Tyr-345 in a single copy of the beta subunit.