To test whether cardioprotection induced by ischemic preconditioning depends on the opening of mitochondrial ATP-sensitive K⁺ (K_ATP) channels, the effect of channel blockade was studied in barbital-anesthetized open-chest pigs subjected to 30 min of complete occlusion of the left anterior descending coronary artery and 3 h of reflow. Preconditioning was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. 5-Hydroxydecanoate (5 mg/kg iv) was injected 15 min before preconditioning or pharmacological preconditioning induced by diazoxide (3.5 mg/kg, 1 ml/min iv). Infarct size (percentage of the area at risk) after 30 min of ischemia was 35.1 ± 9.9% (n = 7). Preconditioning markedly limited myocardial infarct size (2.7 ± 1.6%, n = 7), and 5-hydroxydecanoate did not abolish protection (2.4 ± 0.9%, n = 8). Diazoxide infusion also significantly limited infarct size (14.6 ± 7.4%, n = 7), and 5-hydroxydecanoate blocked this effect (30.8 ± 8.0%, n = 7). Thus the opening of mitochondrial K_ATP channels is cardioprotective in pigs, but these data do not support the hypothesis that opening of mitochondrial K_ATP channels is required for the endogenous protection afforded by preconditioning.