Cytochrome c is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome c release requires a two-step process. Because cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome c involves a breaching of the electrostatic and/or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome c is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome c with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome c release is significantly depressed when the electrostatic interaction between cytochrome c and cardiolipin remains intact. Our results indicate that the release of cytochrome c involves a distinct two-step process that is undermined when either step is compromised.