The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (DeltaPsim) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high DeltaPsim (similar to DeltaPsim in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with DeltaPsim. The application of DeltaPsim up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high DeltaPsim and the increase of FA membrane concentration bring about the significant exponential Gm increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA- remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease DeltaPsim more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by DeltaPsim may be key in mitochondrial respiration and metabolism.