Objective: This study was designed to compare ischemic preconditioning with opening of mitochondrial adenosine triphosphate-sensitive potassium channels and Na+/H+ exchange inhibition in an isolated heart model of cold storage, simulating the situation of cardiac allografts. Methods: Sixty-seven isolated isovolumic buffer-perfused rat hearts were arrested with and stored in Celsior solution (Imtix-Sangstat) at 4°C for 4 hours before a 2-hour reperfusion. Group I hearts served as controls and were arrested with and stored in Celsior solution. In group II, hearts were preconditioned by two 5-minute episodes of global ischemia, each separated by 5 minutes of reperfusion before arrest with Celsior solution. Group III hearts were arrested with and stored in Celsior solution supplemented with 100 μmol/L of the mitochondrial adenosine triphosphate-sensitive potassium channel opener diazoxide. In group IV, hearts received an infusion of diazoxide (30 μmol/L) during the first 15 minutes of reperfusion. Group V hearts underwent a protocol combining both interventions used in groups III and IV. In group VI, hearts were arrested with and stored in Celsior solution supplemented with 1 μmol/L of the Na+/H+ exchange inhibitor cariporide. Group VII hearts received an infusion of cariporide (1 μmol/L) during the first 15 minutes of reperfusion. In group VIII, hearts underwent a protocol combining both interventions used in groups VI and VII. Group IX hearts were ischemically preconditioned as in group II, and sustained Na+/H+ exchange inhibition during both storage and early reperfusion was used as in group VIII. Results: On the basis of comparisons of postischemic left ventricular contractility and diastolic function, coronary flow, total creatine kinase leakage, and myocardial water content, values indicative of improved protection were obtained by combining ischemic preconditioning with Na+/H+ exchange inhibition by cariporide given during storage and initial reperfusion. The endothelium-dependent vasodilatory postischemic responses to 5-hydroxytryptamine or acetylcholine and endothelium-independent responses to papaverine were not affected by these interventions. Conclusions: These data suggest that cardioprotection conferred by the Na+/H+ exchange inhibitor cariporide is additive to that of ischemic preconditioning and might effectively contribute to improve donor heart preservation during cardiac transplantation.