Reactive oxygen species (ROS) production during ischemia/reperfusion (I/R) is thought to be a critical factor for myocardial injury. However a small amount of ROS during the ischemic preconditioning (IPC) may provide a signal for cardioprotection. We have previously reported that the repetitive pretreatment of a small amount of ROS (hydrogen peroxide; H₂O₂, 2 µM) mimicked the IPC-induced cardioprotection in the Langendorff-perfused rat hearts. We further investigated the mechanisms of the ROS-induced cardioprotection against I/R injury, and tested the hypothesis whether it could mediate the mPTP opening. The Langendorff-perfused rat hearts were subjected to 35 min ischemia and 40 min reperfusion, and the pretreatment of H2O2 (2 µM) significantly improved the post-ischemic recoveries in left ventricular developed pressure (LVDP), intracellular phosphocreatine (PCr) and ATP levels. A specific mPTP inhibitor cyclosporin A (CsA; 0.2 µM) canceled these H₂O₂-induced effects. In isolated permeabilized myocytes, H₂O₂ (1 µM) accelerated the calcein leakage from mitochondria in a CsA-sensitive manner, indicating the opening of mPTP by H₂O₂. However, H₂O₂ did not depolarize m even in the presence of oligomycin (F1/F0 ATPase inhibitor; 1 µM), and decreased [Ca²⁺]_m by accelerating the mitochondrial Ca²⁺ extrusion via an mPTP. We conclude that the transient mPTP opening could be involved in the H₂O₂-induced cardioprotection against reperfusion injury, and the reduction of [Ca²⁺]_m without the change in m might be a possible mechanism for the protection.