In order to study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20 min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30 min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30 min ischemic hearts elevated LVEDP, attenuated LVDP and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP/O ratio as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants (N-acetylcysteine or 2-N-mercaptopropionyl glycine). Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical generating system (xanthine plus xanthine oxidase) and an oxidant (H₂O₂) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.