The H + F oF 1-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F 1 moiety of the complex protrudes at the inner side of the membrane, the F o sector spans the membrane reaching the outer side. The IF 1 component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F oF 1-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF 1 (residue 42–58) to the F 1-α/β subunits. We have analyzed the effect of native purified IF 1 the IF 1-(42–58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K +] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F oF 1 complex. The results show that IF 1, and in particular its central 42–58 segment, displays different inhibitory affinity for proton conduction from the F 1 to the F o side and in the opposite direction. Cross-linking of IF 1 to F 1-α/β subunits inhibits the ATP-driven H + translocation but enhances H + conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F oF 1 complex.