We studied the role of mitochondrial ATP-sensitive K+(KATP) channels in modifying functional responses to 20 min global ischemia and 30 min reperfusion in wild-type mouse hearts and in hearts with ~250-fold overexpression of functionally coupled A1-adenosine receptors (A1ARs). In wild-type hearts, time to onset of contracture (TOC) was 303 ᠲ4 s, with a peak contracture of 89 ᠵ mmHg. Diastolic pressure remained elevated at 52 ᠶ mmHg after reperfusion, and developed pressure recovered to 40 ᠶ% of preischemia. A1AR overexpression markedly prolonged TOC to 517 ᠸ4 s, reduced contracture to 64 ᠶ mmHg, and improved recovery of diastolic (to 9 ᠴ mmHg) and developed pressure (to 82 ᠸ%). 5-Hydroxydecanoate (5-HD; 100 卩, a mitochondrial KATPblocker, did not alter ischemic contracture in wild-type hearts, but increased diastolic pressure to 69 ᠸ mmHg and reduced developed pressure to 10 ᠵ% during reperfusion. In transgenic hearts, 5-HD reduced TOC to 348 ᠱ8 s, increased postischemic contracture to 53 ᠴ mmHg, and reduced recovery of developed pressure to 22 ᠴ%. In summary, these data are the first to demonstrate that endogenous activation of KATP channels improves tolerance to ischemia-reperfusion in murine myocardium. This functional protection occurs without modification of ischemic contracture. The data also support a role for mitochondrial KATP channel activation in the pronounced cardioprotection afforded by overexpression of myocardial A1ARs.