The present study was performed to test whether the ischemic preconditioning could reduce mitochondrial O2.- production and prevent mitochondrial respiratory impairment upon reperfusion of ischemic hearts. The isolated perfused rat hearts were subjected to 30 min of global ischemia and 20 min of reperfusion. Ischemic preconditioning was performed, involving three 5-min periods of ischemia, each followed by a 5-min reperfusion just before a sustained ischemia. Ischemic preconditioning improved the post-ischemic cardiac function and reduced LDH release and malondialdehyde production upon reperfusion. 02.- generation of mitochondria isolated from the preconditioned hearts was significantly lower than that of mitochondria from the non-preconditioned hearts, and none of the activities of mitochondrial antioxidant enzymes (SOD, catalase, glutathione peroxidase) was altered as a consequence of the ischemic preconditioning alone. The impairment of mitochondrial state 3 respiration induced by ischemia and reperfusion was prevented by ischemic preconditioning. Amytal, a reversible respiratory chain blocker suppressing 02.- production in mitochondria, prevented the ischemia/reperfusion injury. The cardioprotective effect of Amytal could not be distinguished from that of ischemic preconditioning. These results suggest that the cardioprotective effect of ischemic preconditioning against the ischemia/reperfusion injury is attributed partly to the reduction of mitochondrial oxygen radical generation and prevention of the respiratory impairment during ischemia and reperfusion.