Mitochondrial ATP-sensitive K (mitoK(ATP)) channels play a central role in protecting the heart from injury in ischemic preconditioning. In isolated mitochondria exposed to elevated extramitochondrial Ca, P(i), and anoxia to simulate ischemic conditions, the selective mitoK(ATP) channel agonist diazoxide (25-50 microM) potently reduced mitochondrial injury by preventing both the mitochondrial permeability transition (MPT) and cytochrome c loss from the intermembrane space. Both effects were blocked completely by the selective mitoK(ATP) antagonist 5-hydroxydecanoate. The protective effect against Ca-induced MPT was most evident under conditions in which the ability of electron transport to support membrane potential (Deltapsi(m)) was decreased and inner membrane leakiness was increased moderately. Under these conditions, mitoK(ATP) channel activity strongly regulated Deltapsi(m), and diazoxide prevented MPT by inhibiting the driving force for Ca uptake. Phorbol 12-myristate 13-acetate mimicked the protective effects of diazoxide, unless 5-hydroxydecanoate was present, indicating that protein kinase C activation also protects mitochondria by activating mitoK(ATP) channels. Because Deltapsi(m) recovery ultimately is required for heart functional recovery, these results may explain how mitoK(ATP) channel activation mimics ischemic preconditioning by protecting mitochondria as they pass through a critical vulnerability window during ischemia/reperfusion.