A new property of the presequence of the mitochondrial precursor protein cytochrome oxidase subunit IV is presented. This mitochondrial presequence induces interbilayer contacts between large unilamellar vesicles consisting of phosphatidylcholine and cardiolipin. The presequence-vesicle aggregates can be dissociated by applying a membrane potential across the bilayers (negative inside). These effects require the presence of cardiolipin and are not observed for other negatively charged phospholipids. We propose a role for the presequence in the formation and dissociation of mitochondrial contact sites.