Oxygen radicals and protein kinase C (PKC) mediate ischemic preconditioning. Using a cultured chick embryonic cardiomyocyte model of hypoxia and reoxygenation, we found that the oxygen radicals generated by ischemic preconditioning were H₂O₂. Like preconditioning, H₂O₂ selectively activated the ε-isoform of PKC in the particulate compartment and increased cell viability after 1 h of hypoxia and 3 h of reoxygenation. The glutathione peroxidase ebselen (converting H₂O₂ to H₂O) and the superoxide dismutase inhibitor diethyldithiocarbamic acid abolished the increased H₂O₂ and the protection of preconditioning. PKC activation with phorbol 12-myristate 13-acetate increased cell survival; the protection of preconditioning was blocked by εV_1–2, a selective PKC-ε antagonist. Similar to preconditioning, the protection of PKC activation was abolished by mitochondrial K_ATP channel blockade with 5-hydroxydecanoate or by GABA receptor stimulation with midazolam or diazepam. In addition, PKC, mitochondrial ATP-sensitive K⁺(K_ATP) channels, and GABA receptors had no effects on H₂O₂ generated by ischemic preconditioning before prolonged hypoxia and reoxygenation. We conclude that H₂O₂ opens mitochondrial K_ATPchannels and inhibits GABA receptors via activating PKC-ε. Through this signal transduction, preconditioning protects ischemic cardiomyocytes.