Restoration of the blood flow after a period of ischemia is accompanied by generation of toxic oxygen radicals. This phenomenon may account for the occurrence of reperfusion-mediated tissue injury in ischemic hearts. In in vitro studies, although oxygen radicals can be generated from a variety of sources, including xanthine oxidase system, activated leucocytes, mitochondria and others, the most important source and mechanism of oxygen radical production in the post-ischemic reperfused hearts is unclear. In the present study, we tested the hypothesis that the respiratory chain of mitochondria might be an important source of oxygen radicals which are responsible for the development of the reperfusion injury of ischemic hearts. Langendorff-perfused, isolated rat hearts were subjected to 30 min of global ischemia at 37˚C, followed by reperfusion. Amytal, a reversible inhibitor of mitochondrial respiration, was employed to assess the mitochondrial contributions to the development of the reperfusion injury. Intact mitochonria were isolated from the control and the post-ischemic reperfused hearts. Mitochondrial oxygen radical generation was measured by chemiluminescence method and the oxidative tissue damage was estimated by measuring a lipid peroxidation product, malondialdehyde(MDA). To evaluate the extent of the reperfusion injury, post-ischemic functional recovery and lactate dehydrogenase(LDH) release were assessed and compared in Amytal-treated and -untreated hearts. Upon reperfusion of the ischemic hearts, MDA release into the coronary effluent was markedly increased. MDA content of mitochondria isolated from the post-ischemic reperfused hearts was increased to 152% of preischemic value, whereas minimal change was observed in extramitochondrial fraction. The generation of superoxide anion was increased about twice in mitochondria from the reperfused hearts than in those from the control hearts. Amytal inhibited the mitochondrial superoxide generation significantly and also suppressed MDA production in the reperfused hearts. Additionally, Amytal prevented the contractile dysfunction and the increased release of LDH observed in the reperfused hearts. In conclusion, these results indicate that the respiratory chain of mitochondria may be an important source of oxygen radical formation in post-ischemic reperfused hearts, and that oxygen radicals originating from the mitochondria may contribute to the development of myocardial reperfusion injury.