Pharmacological evidence has implicated ATP-dependent potassium (KATP) channels in the mechanism of ischemic preconditioning; however, the effects of sarcolemmal KATP channels on excitability cannot account for the protection. KATP channels also exist in mitochondrial inner membrane. To test whether such channels play a role in cardioprotection, we simultaneously measured flavoprotein fluorescence, an index of mitochondrial redox state, and sarcolemmal KATP currents in intact rabbit ventricular myocytes. Our results show that diazoxide, a KATP channel opener, induced reversible oxidation of flavoproteins, but did not activate sarcolemmal KATP channels. This effect of diazoxide was blocked by 5-hydroxydecanoic acid (5-HD). We further verified that 5-HD is a selective blocker of the mitochondrial KATP channels. These methods have enabled us to demonstrate that the activity of mitochondrial KATP channels can be regulated by protein kinase C. In a cellular model of simulated ischemia, inclusion of diazoxide decreased the rate of cell death to about half of that in control. Such protection is inhibited by 5-HD. In conclusion, our results demonstrate that diazoxide targets mitochondrial but not sarcolemmal KATP channels, and imply that mitochondrial KATP channels may mediate preconditioning.