Reactive oxygen species (ROS) contribute to cell damage during reperfusion of the heart. ROS may exert their effects partly by interfering with Ca 2 + homeostasis of the myocardium. The purpose of this study was to investigate the effects of hydrogen peroxide (H 2 O 2 ) on Ca 2 + accumulation during reoxygenation of isolated adult rat cardiomyocytes exposed to 1 h of hypoxia and to relate the effects to possible changes in release of lactate dehydrogenase (LDH), free intracellular Ca 2 + ([Ca 2 + ] i ) and Mg 2 + ([Mg 2 + ] i ), and mitochondrial membrane potential (Δψm). Cell Ca 2 + was determined by 4 5 Ca 2 + uptake. Free [Mg 2 + ] i and [Ca 2 + ] i and Δψm were measured by flow cytometry. Reoxygenation-induced Ca 2 + accumulation was attenuated by 23 and 34% by 10 and 25 μM H 2 O 2 , respectively, added at reoxygenation. H 2 O 2 at 100 and 250 μM increased cell Ca 2 + by 50 and 83%, respectively, whereas 500 μM H 2 O 2 decreased cell Ca 2 + by 20%. H 2 O 2 at (25 μM) reduced LDH release and [Mg 2 + ] i and increased Δψm, indicating cell protection, whereas 250 μM H 2 O 2 increased LDH release and [Mg 2 + ] i and decreased Δψm, indicating cell damage. Clonazepam (100 μM) attenuated the increase in Ca 2 + accumulation, the elevation of [Ca 2 + ] i , and the decrease in Δψm induced by 100 and 250 μM H 2 O 2 during reoxygenation. We report for the first time that 25 μM H 2 O 2 attenuates Ca 2 + accumulation, LDH release, and dissipation of Δψm during reoxygenation of hypoxic cardiomyocytes, indicating cell protection.