Objective: We propose that ischemic preconditioning (IPC) and mitochondrial K_ATP channel activation protect the myocardium by inhibiting mitochondrial permeability transition pore (MPTP) opening at reperfusion. Methods: Isolated rat hearts were subjected to 35 min ischemia/120 min reperfusion and assigned to the following groups: (1) control; (2) IPC of 2×5 min each of preceding global ischemia; (3,4,5) 0.2 μmol/l cyclosporin A (CsA, which inhibits MPTP opening), 5 μmol/l FK506 (which inhibits the phosphatase calcineurin without inhibiting MPTP opening), or 20 μmol/l atractyloside (Atr, a MPTP opener) given at reperfusion; (6,7) pre-treatment with 30 μmol/l diazoxide (Diaz, a mitochondrial K_ATP channel opener) or 200 nmol/l 2 chloro-N⁶-cyclopentyl-adenosine (CCPA, an adenosine A1 receptor agonist); (8) IPC+Atr; (9) Diaz+Atr; (10) CCPA+Atr. The effect of mitochondrial K_ATP channel activation on calcium-induced MPTP opening in isolated calcein-loaded mitochondria was also assessed. Results: IPC, CsA when given at reperfusion, and pre-treatment with diazoxide or CCPA all limited infarct size (19.9±2.6% in IPC; 24.6±1.9% in CsA, 18.0±1.7% in Diaz, 20.4±3.3% in CCPA vs. 44.7±2.0% in control, P<0.0001). Opening the MPTP with atractyloside at reperfusion abolished this cardio-protective effect (47.7±1.8% in IPC+Atr, 42.3±3.2% in Diaz+Atr, 51.2±1.6% in CCPA+Atr). Atractyloside and FK506, given at reperfusion, did not influence infarct size (45.7±2.1% in Atr and 43.1±3.6% in FK506 vs. 44.7±2.0% in control, P = NS). Diazoxide (30 μmol/l) was shown to reduce calcium-induced MPTP opening by 52.5±8.0% in calcein-loaded mitochondria. 5-Hydroxydecanoic acid (100 μmol/l) was able to abolish the cardio-protective effects of both diazoxide and IPC. Conclusion: One interpretation of these data is that IPC and mitochondrial K_ATP channel activation may protect the myocardium by inhibiting MPTP opening at reperfusion.