R ecently it has been postulated that mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels rather than sarcolemmal K_ATP (sarcK_ATP) channels are important as end effectors and/or triggers of ischemic preconditioning (IPC). To define the pathophysiological significance of sarcK_ATP channels, we conducted functional experiments using Kir6.2-deficient (KO) mice. Metabolic inhibition with glucose-free, dinitrophenol-containing solution activated sarcK_ATP current and shortened the action potential duration in ventricular cells isolated from wild-type (WT) but not KO mice. MitoK_ATP channel function was preserved in KO ventricular cells. In anesthetized mice, IPC reduced the infarct size in WT but not KO mice. Following global ischemia/reperfusion, the increase of left ventricular end-diastolic pressure during ischemia was more marked, and the recovery of contractile function was worse, in KO hearts than in WT hearts. Treatment with HMR1098, a sarcK_ATP channel blocker, but not 5-hydroxydecanoate, a mitoK_ATP channel blocker, produced a deterioration of contractile function in WT hearts comparable to that of KO hearts. These findings suggest that sarcKATP channels figures prominently in modulating ischemia/reperfusion injury in the mouse. The rapid heart rate of the mouse (>600 beats per minute) may magnify the relative importance of sarcK_ATP channels during ischemia, prompting caution in the extrapolation of the conclusions to larger mammals.