Inhibition of the yeast F(0)F(1)-ATP synthase by the regulatory peptides IF1 and STF1 was studied using intact mitochondria and submitochondrial particles from wild-type cells or from mutants lacking one or both peptides. In intact mitochondria, endogenous IF1 only inhibited uncoupled ATP hydrolysis and endogenous STF1 had no effect. Addition of alamethicin to mitochondria readily made the mitochondrial membranes permeable to nucleotides, and bypassed the kinetic control exerted on ATP hydrolysis by the substrate carriers. In addition, alamethicin made the regulatory peptides able to cross mitochondrial membranes. At pH 7.3, F(0)F(1)-ATPase, initially inactivated by either endogenous IF1 or endogenous STF1, was completely reactivated hours or minutes after alamethicin addition, respectively. Previous application of a membrane potential favored the release of endogenous IF1 and STF1. These observations showed that IF1 and STF1 can fully inhibit ATP hydrolysis at physiological concentrations and are sensitive to the same effectors. However, ATP synthase has a much lower affinity for STF1 than for IF1, as demonstrated by kinetic studies of ATPase inhibition in submitochondrial particles by externally added IF1 and STF1 at pHs ranging from 5.5 to 8.0. Our data do not support previously proposed effects of STF1, like the stabilization of the IF1-F(0)F(1) complex or the replacement of IF1 on its binding site in the presence of the proton-motive force or at high pH, and raise the question of the conditions under which STF1 could regulate ATPase activity in vivo.