Increased resistance to myocardial ischemia in chronically hypoxic immature rabbit hearts is associated with activation of ATP-sensitive K+(KATP) channels. We determined whether chronic hypoxia from birth alters the function of the mitochondrial KATPchannel. The KATPchannel opener bimakalim (1 &mgr;mol/L) increased postischemic recovery of left ventricular developed pressure in isolated normoxic (Fio2=0.21) hearts to values (42±4% to 67±5% ) not different from those of hypoxic controls but did not alter postischemic recovery of developed pressure in isolated chronically hypoxic (Fio2=0.12) hearts (69±5% to 72±5%). Conversely, the KATPchannel blockers glibenclamide (1 &mgr;mol/L) and 5-hydroxydecanoate (5-HD, 300 &mgr;mol/L) attenuated the cardioprotective effect of hypoxia but had no effect on postischemic recovery of function in normoxic hearts. ATP synthesis rates in hypoxic heart mitochondria (3.92±0.23 &mgr;mol ATP · min−1· mg mitochondrial protein−1) were significantly greater than rates in normoxic hearts (2.95±0.08 &mgr;mol ATP · min−1· mg mitochondrial protein−1). Bimakalim (1 &mgr;mol/L) decreased the rate of ATP synthesis in normoxic heart mitochondria consistent with mitochondrial KATPchannel activation and mitochondrial depolarization. The effect of bimakalim on ATP synthesis was antagonized by the KATPchannel blockers glibenclamide (1 &mgr;mol/L) and 5-HD (300 &mgr;mol/L) in normoxic heart mitochondria, whereas glibenclamide and 5-HD alone had no effect. In hypoxic heart mitochondria, the rate of ATP synthesis was not affected by bimakalim but was attenuated by glibenclamide and 5-HD. We conclude that mitochondrial KATPchannels are activated in chronically hypoxic rabbit hearts and implicate activation of this channel in the improved mitochondrial bioenergetics and cardioprotection observed.