From in vivo models of stroke it is known that ischemia/reperfusion induces oxidative stress that is accompanied by deterioration of brain mitochondria. Previously, we reported that the increase in Ca2+ induces functional breakdown and morphological disintegration in brain mitochondria subjected to hypoxia/reoxygenation (H/R). Protection by ADP indicated the involvement of the mitochondrial permeability transition pore in the mechanism of membrane permeabilization. Until now it has been unclear how reactive oxygen species (ROS) contribute to this process. We now report that brain mitochondria which had been subjected to H/R in the presence of low micromolar Ca2+ display low state 3 respiration (20% of control), loss of cytochrome c, and reduced glutathione levels (75% of control). During reoxygenation, significant mitochondrial generation of hydrogen peroxide (H2O2) was detected. The addition of the membrane permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) suppressed the production of H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions. TEMPOL partially protected brain mitochondria exposed to H/R and low micromolar Ca2+ from decrease in state 3 respiration (from 25% of control to 60% of control with TEMPOL) and permeabilization of the inner membrane. Membrane permeabilization was obvious, because state 3 respiration could be stimulated by extramitochondrial NADH. Our data suggest that ROS and Ca2+ synergistically induce permeabilization of the inner membrane of brain mitochondria exposed to H/R. However, permeabilization can only partially be prevented by suppressing mitochondrial generation of ROS. We conclude that transient deprivation of oxygen and glucose during temporary ischemia coupled with elevation in cytosolic Ca2+ concentration triggers ROS generation and mitochondrial permeabilization, resulting in neural cell death.