Background - Recent studies have implicated mitochondrial ATP-sensitive potassium (K(ATP)) channels in the cardioprotective effects of ischemic preconditioning. The present study used a model of prolonged cold heart storage to assess whether the mitochondrial K(ATP) opener diazoxide could reproduce the protection conferred by ischemic preconditioning. Methods and Results - Fifty-four isolated rat hearts were arrested with and stored in Celsior at 4°C for 10 hours before a 2-hour reperfusion. They were divided into 5 groups. Group 1 hearts served as controls. In group 2, hearts were preconditioned by two 5-minute episodes of global ischemia, each separated by 5 minutes of reperfusion before arrest. In group 3, hearts received a 15- minute infusion of the mitochondrial K(ATP) opener diazoxide (30 μmol/L) followed by 5 minutes of washout before arrest. In groups 4 and 5, hearts underwent a protocol similar to that used in groups 2 and 3, respectively, except that the preconditioning was preceded by a 10-minute infusion of the mitochondrial K(ATP) blocker 5-hydroxydecanoate (5-HD, 100 μmol/L). Both ischemic and diazoxide preconditioning provided a similar degree of cardioprotection demonstrated by a significantly better preservation of left ventricular compliance, reduced leakage of creatine kinase, and smaller degree of myocardial edema compared with control hearts. These beneficial effects were abolished by 5-HD pretreatment. Postischemic left ventricular contractility and endothelium-dependent coronary response to 5- hydroxytryptamine and acetylcholine were not different among groups. However, the endothelium-independent vasodilatory postischemic response to papaverine was better preserved after ischemic and diazoxide preconditioning than in the other groups. Conclusions - These data support the concept that the cardioprotective effects of ischemic preconditioning can be duplicated by a mitochondrial K(ATP) opener and suggest that activation of these channels could be an effective means of improving the preservation of globally ischemic cold-stored hearts, as occurs during cardiac transplantation.