Digitonin-permeabilized PC12 and GT1-7 neural cells exhibited a cyclosporin A-sensitive decrease in mitochondrial membrane potential, increased volume, and release of the pro-apoptotic factor cytochrome c in the presence of Ca2+ and the mitochondrial permeability transition (MPT) inducers t-butyl hydroperoxide (t-bOOH) or phenylarsine oxide (PhAsO). Although the concentration of PhAsO required to induce the MPT was similar for Bcl-2 negative and Bcl-2 overexpressing transfected cells (Bcl-2(+)), the level of t-bOOH necessary for triggering the MPT was much higher for Bcl-2(+) cells. A higher concentration of t-bOOH was also necessary for promoting the oxidation of mitochondrial pyridine nucleotides in Bcl-2(+) cells. The sensitivity of Bcl-2(- ) cell mitochondria to t-bOOH but not PhAsO could be overcome by the use of conditions that protect the pyridine nucleotides against oxidation. We conclude that the increased ability of Bcl-2(+) cells to maintain mitochondrial pyridine nucleotides in a reduced redox state is a sufficient explanation for their resistance to MPT under conditions of oxidative stress induced by Ca2+ plus t-bOOH.