The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pHout) of PCPECL liposomes, with an internal pH (pHin) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK a ~ 6.9). Conversely, ΔpH generated by enhanced pHin of PCPECL at a pHout of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pHin at a pHout of 8.0. At bulk acidic pH, ΔΨ generated by Na+ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pHout, the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨM blocks inner mitochondrial membrane fusion during apoptosis.