Anoxia/reoxygenation injury of isolated rat liver mitochondria was investigated. During anoxia of up to 60 min, the membrane potential was largely preserved and mitochondrial swelling was not observed. Reoxygenation of anoxic mitochondria rapidly caused swelling, cyclosporin A-sensitive Ca2+ efflux, [14C]sucrose trapping, and loss of the membrane potential along with increased generation of reactive oxygen intermediates (ROI). Although pretreatment with catalase and superoxide dismutase completely abolished reoxygenation-induced generation of ROI, mitochondrial damage was not prevented, as indicated by swelling, loss of the membrane potential, a decrease of the ATP content, and cyclosporin A-sensitive Ca2+ efflux. However, addition of the immunosuppressant cyclosporin A or addition of ADP completely prevented the mitochondrial damage induced by reoxygenation. The same protective effect was noted when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with ruthenium red. These findings indicate that mitochondrial anoxia/reoxygenation injury is caused by the cyclosporin A-sensitive and Ca2+-dependent membrane permeability transition. In contrast, reoxygenation injury does not appear to be triggered by the enhanced production of ROI.