Cytochrome c release from mitochondria during apoptosis is a critical step, but the precise mechanisms remain unclear. This study demonstrates that cytochrome c release from isolated liver mitochondria involves a two-step process. First, the electrostatic and/or hydrophobic interactions between cytochrome c and cardiolipin must be disrupted to solubilize the protein. Second, permeabilization of the outer mitochondrial membrane by Bax is required to release the solubilized cytochrome c into the extracellular environment. Neither step alone is sufficient to trigger cytochrome c release. The study also shows that oxidative stress can mobilize a unique pool of tightly bound cytochrome c by peroxidating cardiolipin, further supporting the two-step nature of cytochrome c release. These findings provide insights into the mechanisms underlying cytochrome c release during apoptosis.Cytochrome c release from mitochondria during apoptosis is a critical step, but the precise mechanisms remain unclear. This study demonstrates that cytochrome c release from isolated liver mitochondria involves a two-step process. First, the electrostatic and/or hydrophobic interactions between cytochrome c and cardiolipin must be disrupted to solubilize the protein. Second, permeabilization of the outer mitochondrial membrane by Bax is required to release the solubilized cytochrome c into the extracellular environment. Neither step alone is sufficient to trigger cytochrome c release. The study also shows that oxidative stress can mobilize a unique pool of tightly bound cytochrome c by peroxidating cardiolipin, further supporting the two-step nature of cytochrome c release. These findings provide insights into the mechanisms underlying cytochrome c release during apoptosis.