The interaction of peroxidized cardiolipin with ferrocytochrome c induces two kinetically and chemically distinct processes. The first is a rapid oxidation of ferrocytochrome c, followed by a slower, irreversible disruption of heme c. The oxidation of ferrocytochrome c by peroxidized cardiolipin is explained by a Fenton-type reaction. Heme scission is a consequence of the radical-mediated reactions initiated by the interaction of ferric heme iron with peroxidized cardiolipin. Simultaneously with the heme c disruption, generation of hydroxyl radical is detected by EPR spectroscopy using the spin trapping technique. The resulting apocytochrome c sediments as a heterogeneous mixture of high aggregates, as judged by sedimentation analysis. Both the oxidative process and the destructive process were suppressed by nonionic detergents and/or high ionic strength. The mechanism for generating radicals and heme rupture is presented.