Mitochondrial permeability transition is typically characterized by Ca2+ and oxidative stress-induced opening of a nonselective proteinaceous membrane pore sensitive to cyclosporin A, known as the permeability transition pore (PTP). Data from our laboratory provide evidence that the PTP is formed when inner membrane proteins aggregate as a result of disulfide cross-linking caused by thiol oxidation. Here we compared the redox properties between PTP in intact mitochondria and mitoplasts. The rat liver mitoplasts retained less than 5% and 10% of the original outer membrane markers monoamine oxidase and VDAC, respectively. Kidney mitoplasts also showed a partial depletion of hexokinase. In line with the redox nature of the PTP, mitoplasts that were more susceptible to PTP opening than intact mitochondria showed higher rates of H2O2 generation and decreased matrix NADPH-dependent antioxidant activity. Mitoplast PTP was also sensitive to the permeability transition inducer tert-butyl hydroperoxide and to the inhibitors cyclosporin A, EGTA, ADP, dithiothreitol and catalase. Taken together, these data indicate that, in mitoplasts, PTP exhibits redox regulatory characteristics similar to those described for intact mitochondria.