—Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na⁺-H⁺ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 μmol/L hydrogen peroxide (H₂O₂) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H₂O₂ (100 μmol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H₂O₂. Pretreatment with chelerythrine (10 μmol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 μmol/L, 45 minutes) or herbimycin A (3 μmol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H₂O₂-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H₂O₂ also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 μmol/L, 60 minutes). These results suggest that low doses of H₂O₂ induce MAP kinase–dependent pathways that regulate NHE activity during reperfusion injury.