Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca2+concentration ([Ca2+]m), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoKATPchannel opener diazoxide attenuated the accumulation of [Ca2+]mduring simulated I/R (EC50=18 &mgr;mol/L). These effects of diazoxide were blocked by the mitoKATPchannel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca2+]maccumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, &Dgr;&PSgr;m, in permeabilized myocytes revealed that diazoxide depolarized &Dgr;&PSgr;m(by 12% at 10 &mgr;mol/L,P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca2+overload, as a consequence of partial mitochondrial membrane depolarization by mitoKATPchannels, underlies cardioprotection. Furthermore, mitoKATPchannels and the MPT differentially affect mitochondrial calcium homeostasis: mitoKATPchannels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion.