Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C ε (PKCε) has been identified as a component of the mitoK_ATP signaling cascade. We hypothesized that PKCε and mitoK_ATP interact directly to form functional signaling modules in the inner mitochondria membrane. To examine this possibility, we studied K⁺ flux in liposomes containing partially purified mitoK_ATP. The reconstituted proteins were obtained after detergent extraction of isolated mitochondria, 200-fold purification by ion exchange chromatography, and reconstitution into lipid vesicles. Immunoblot analysis revealed the presence of PKCε in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKCε peptide agonist, ψεRACK, each activated mitoK_ATP-dependent K⁺ flux in the reconstituted system. This effect of PKCε was prevented by chelerythrine, by the specific PKCε peptide antagonist, εV_1-2, and by the specific mitoK_ATP inhibitor 5-hydroxydecanoate. In addition, the activating effect of PKC agonists was reversed by exogenous protein phosphatase 2A. These results demonstrate persistent, functional association of mitochondrial PKCε and mitoK_ATP.