Overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in mitochondria of RBL2H3 cells (M15 cells) prevented the release of cytochrome c (cyt. c), the activation of caspase-3, and apoptosis caused by 2-deoxyglucose (2DG), whereas cells overexpressing nonmitochondrial PHGPx(L9) and control (S1) cells were induced to apoptosis. Hydroperoxide levels in mitochondria of L9 and S1 cells were significantly enhanced by 2DG-induced apoptosis. In contrast, generation of hydroperoxide in mitochondria was protected in M15 cells, which also showed resistance to apoptosis by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, stimuli that induce apoptosis by the liberation of cyt. c from mitochondria. Cyt.c preferentially binds to the monolayer of cardiolipin (CL), the specific phospholipid of the inner membrane of mitochondria. The amount of cyt. c bound to the monolayer of cardiolipin hydroperoxide (CL-OOH) was much lower than that bound to CL. Cytc bound to liposome containing CL was released by peroxidation with a radical initiator. Adenine nucleotide translocator (ANT), which regulates the opening and closing the permeability transition (PT) pore, potentially was inactivated in apoptosis-induced S1 cells 4 h after the addition of 2DG, coincidentally with cyt. c release from mitochondria. ANT activity was suppressed by the fusion of isolated mitochondria with liposomes containing CL-OOH. ANT activity was expressed in proteoliposomes containing 10% CL, but it was competitively inhibited by the addition of CL-OOH. This study suggests that CL peroxidation might have an initiating role in the liberation of cyt. c from the inner membrane, and in the opening of the PT pore via inactivation of ANT. Mitochondrial PHGPx might play a role as an anti-apoptotic factor by protecting CL and reducing CL-OOH.