Objective: The present study aimed to elucidate the involvement of sodium overload and following damage to mitochondria during ischemia in the genesis of ischemia/reperfusion injury of perfused rat hearts. Methods: Isolated, perfused hearts were exposed to different durations (15–35 min) of ischemia followed by 60-min reperfusion. At the end of ischemia or reperfusion, myocardial sodium and calcium contents and myocardial high-energy phosphates were determined. The cardiac mitochondrial ability to produce ATP was measured using saponin-skinned bundles. The effects of sodium on the mitochondrial membrane potential and the oxidative phosphorylation rate were examined using isolated mitochondria from normal hearts. Results: Post-ischemic recovery of left ventricular developed pressure decreased in an ischemic duration-dependent manner. Ischemia induced an increase in myocardial sodium, but not calcium. This increase was dependent on the duration of ischemia. The oxygen consumption rate of skinned bundles from the ischemic heart decreased at the end of ischemia. Incubation of mitochondria with various concentrations of sodium chloride or sodium lactate in vitro resulted in a depolarization of mitochondrial membrane potential and a decrease in ATP-generating activity. This decrease was not restored after elimination of sodium compounds. Conclusions: The present findings suggest that ischemia induces an increase in sodium influx from the extracellular space and that accumulated sodium may induce irreversible damage to mitochondria during ischemia. This mitochondrial dysfunction may be one of the most important determinants for the genesis of ischemia/reperfusion injury in perfused rat hearts.