We studied the role of mitochondrial ATP-sensitive K⁺(K_ATP) 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 ± 24 s, with a peak contracture of 89 ± 5 mmHg. Diastolic pressure remained elevated at 52 ± 6 mmHg after reperfusion, and developed pressure recovered to 40 ± 6% of preischemia. A₁AR overexpression markedly prolonged TOC to 517 ± 84 s, reduced contracture to 64 ± 6 mmHg, and improved recovery of diastolic (to 9 ± 4 mmHg) and developed pressure (to 82 ± 8%). 5-Hydroxydecanoate (5-HD; 100 μM), a mitochondrial K_ATPblocker, did not alter ischemic contracture in wild-type hearts, but increased diastolic pressure to 69 ± 8 mmHg and reduced developed pressure to 10 ± 5% during reperfusion. In transgenic hearts, 5-HD reduced TOC to 348 ± 18 s, increased postischemic contracture to 53 ± 4 mmHg, and reduced recovery of developed pressure to 22 ± 4%. In summary, these data are the first to demonstrate that endogenous activation of K_ATP 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 K_ATP channel activation in the pronounced cardioprotection afforded by overexpression of myocardial A₁ARs.