Impairment of mitochondrial function is a central event of ischemia-reperfusion (I/R) injury leading to tissue damage and cell death. We studied the relationship between mitochondrial Ca2+ overload, reactive oxygen species (ROS) generation, opening of the mitochondrial permeability transition pore (mPTP) and cell death in rabbit ventricular myocytes exposed to simulated I/R. Changes in mitochondrial Ca2+ ([Ca2+]m) were measured with X-Rhod-1, ROS generation with Mito-Sox Red, mPTP opening as mitochondrial calcein red release, and cell death as lactate dehydrogenase release. I/R was induced by exposing cells to glucose-free Tyrode solution containing 20 mM 2-deoxyglucose and 2 mM NaCN, pH 6.4, followed by superfusion with standard Tyrode solution. No cell death was observed after 20 min of ischemia despite a significant increase in [Ca2+]m, ROS and mPTP opening, however cell death increased significantly after 15 min of reperfusion. The Ca2+ uniporter blocker Ru360 partially prevented [Ca2+]m increase and completely abolished ROS generation and cell death when applied during I/R, however application of Ru360 only during reperfusion did not protect from cell death. Scavenging ROS with superoxide dismutase mimetic MnTBAP or antioxidant Trolox prevented reperfusion-induced cell death. Blocking mPTP during ischemia by cyclosporine A or depletion of mitochondrial inorganic polyphosphate did not provide protection against cell death during reperfusion, but instead led to increased mitochondrial ROS accumulation. However, inhibiting mPTP opening during reperfusion with cyclosporine A attenuated cell death. We conclude that mPTP-dependent cell death during reperfusion is mediated by Ca2+-dependent ROS generation during ischemia. Moreover, our data suggest that mPTP opening during ischemia could serve as ROS escape pathway from mitochondria and thereby attenuate mitochondrial ROS accumulation and ROS-mediated cell damage during subsequent reperfusion.