Background Recent studies have implicated the opening of mitochondrial K_ATP (mitoK_ATP) channels and the production of reactive oxygen species (ROS) in the cardioprotective mechanism of ischemic preconditioning (IPC). Methods and Results The involvement of mitoK_ATP channels and ROS in the cardioprotective effects of both IPC and the mitoK_ATP channel opener diazoxide (DZ) was investigated in ischemic/reperfused rat hearts. The effects of IPC and DZ on myocardial high-energy phosphate concentrations and intracellular pH (pH_i) were also examined using ³¹P nuclear magnetic resonance spectroscopy. Although both the mitoK_ATP channel inhibitor 5-hydroxydecanoate and the antioxidant N-acetylcysteine abolished the postischemic recovery of contractile function by DZ, neither of them inhibited that by IPC. IPC attenuated the decline in pHi during ischemia, but DZ did not (6.28±0.04 in IPC, p<0.05, and 6.02±0.05 in DZ vs 6.02 ±0.06 in control hearts). DZ, but not IPC, reduced the decrease in ATP levels during ischemia (ATP levels at 20-min ischemia: 26.3±3.4% of initial value in DZ, p<0.05, and 8.1±3.0% in IPC vs 15.1±1.3% in control hearts). Conclusions These results suggest that DZ-induced cardioprotection is related to ROS production and reduced ATP degradation during ischemia, whereas attenuated acidification during ischemia is involved in IPC-induced cardioprotection, which is not mediated through mitoK_ATP channel opening or ROS production. (Circ J 2004; 68: 156 - 162)