It was shown previously that the ATP synthase complex of bovine heart mitochondria contains an essential set of thiols or dithiols in its membrane sector (F0), whose modification by various reagents results in uncoupling [Yagi, T., and Hatefi, Y. (1984) Biochemistry 23, 2449-2455]. The sensitivity to modifiers was increased by membrane energization, and the uncoupling was reversed by membrane-permeable thiol compounds when modifiers other than alkylating agents were used to uncouple. The present paper demonstrates that there exists in the F0 of bovine ATP synthase another set of essential thiols, whose modification results in reversible inhibition of ATPase activity. These thiols are most susceptible to modification by mercurials (p-chloromercuribenzoate greater than p-chloromercuribenzene sulfonate) and do not appear to be modified by N-ethylmaleimide. The reversible modification of these thiols by mercurials protects the ATP synthase against irreversible inhibition in F0 by N,N-dicyclohexylcarbodiimide. The possible location of these two sets of thiols in the F0 of bovine ATP synthase is discussed.