We examined the effect of cardioprotective agents on three distinct phases of the H₂O₂-induced response that leads to loss of mitochondrial membrane potential (ΔΨ_m) and cell death in cultured cardiac myocytes: (1) priming, consisting of calcium-dependent morphological changes in mitochondria (swelling and loss of cristae), with preserved ΔΨ_m, (2) depolarization, the rapid ΔΨ_m depolarization caused by mitochondrial permeability transition pore (PTP) opening, and (3) cell fragmentation. The mitochondrial ATP-sensitive potassium (mitoK_ATP) channel opener diazoxide markedly decreased the likelihood that cells would undergo priming: many mitochondria remained fully polarized and morphologically intact. Diazoxide not only decreased the number of cells undergoing ΔΨ_m depolarization but also delayed the onset of ΔΨ_m loss, whereas it did not change the duration of depolarization in unprotected cells. The adenine nucleotide translocase inhibitor bongkrekic acid mimicked the effect of diazoxide to suppress priming, except that its effects were not blocked by the mitoK_ATP channel blocker 5-hydroxydecanoate. In contrast, the PTP inhibitor cyclosporin A (CsA) did not prevent priming: neither latency for ΔΨ_m depolarization nor mitochondrial morphological changes were affected. However, CsA slowed the process of depolarization and blunted its severity. Importantly, coapplication of diazoxide and CsA exhibited additive effects, improving the efficacy of protection. Activation of mitoK_ATP channels suppresses the cell death process at its earliest stage, by preserving mitochondrial integrity during oxidative stress. By virtue of its pharmacology and its phenotypic consequences, this mode of action is distinguishable from that of other cardioprotective interventions.