Cytochrome c (cyt c), a component of the respiratory chain, promotes apoptosis when released into the cytosol. Cyt c anchorage within mitochondria depends on cardiolipin (CL). Detachment and release have been related to CL loss and peroxidation. We report that NaN(3)-dependent complex IV inhibition, accompanied by impairment of respiration, resulted in cyt c release. Contrarily, inhibition of respiration upstream cyt c with complex I and III inhibitors was not accompanied by the release of the protein, despite CL decrease and monolyso-CL increase. No CL changes and H(2)O(2) formation were observed by inhibiting complex IV. In cyt c-CL liposomes, breaching cyt c-CL hydrophilic interactions produced a higher release of the reduced, compared to the oxidized form, suggesting that the hydrophobic component of cyt c-CL binding is prevalent in the oxidized form. Free or liposome-reconstituted cyt c was able to form fatty acid-protein complexes (palmitate < linoleate < oleate) only in its reduced form. We hypothesize that reduced cyt c-fatty acid binding favors the dislocation of the protein from anchoring CL. A mechanism for cyt c release independent of CL peroxidation by H(2)O(2) is feasible. It could weaken the hydrophobic component of cyt c-CL interactions and might function following complex IV inhibition or in oxygen lack, both conditions producing accumulation of reduced cyt c and free fatty acids.