Previous studies showed a poor correlation between sarcolemmal K⁺ currents and cardioprotection for ATP-sensitive K⁺ channel (K_ATP) openers. Diazoxide is a weak cardiac sarcolemmal K_ATP opener, but it is a potent opener of mitochondrial K_ATP, making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart K_ATP, diazoxide opened mitochondrial K_ATP with a K_1/2 of 0.8 μmol/L while being 1000-fold less potent at opening sarcolemmal K_ATP. To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC₂₅, 11.0 and 8.8 μmol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. Whole-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K⁺ currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac K_ATP activity. Cromakalim and diazoxide were both potent activators of K⁺ flux in this preparation (K_1/2 values, 1.1±0.1 and 0.49±0.05 μmol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K⁺ flux through the diazoxide-opened mitochondrial K_ATP. The profile of activity of diazoxide (and perhaps K_ATP openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial K_ATP.