We examined the role of intracellular catalase activity in modulating hydrogen peroxide (H2O2)-induced cytotoxicity in cultured chick embryo cardiac myocytes. Injury was quantitated by release of lactate dehydrogenase (LDH). Application of 1.5 mM H2O2 to myocytes caused LDH release beginning at 2 h. Inactivation or inhibition of catalase with aminotriazole or sodium azide increased LDH release but did not cause earlier release. Free catalase which entered or became associated with myocytes, but not catalase bound to agarose beads, which did not enter or become associated with myocytes, was protective. Separate experiments demonstrated that myocyte catalase activity decreased by 27% between 1 and 4 h of H2O2 exposure. Treatment with aprotinin, a protease inhibitor, prevented the H2O2-induced fall in catalase activity at 4 h but treatment with deferoxamine, an iron chelator, had no effect on catalase activity. Thus, with exposure of cardiac myocytes to H2O2, the magnitude of the cytotoxicity is modulated by endogenous or cell associated exogenous catalase. It is proposed that in addition to excessive accumulation of H2O2, a reduction intracellular catalase activity may be required before substantial cell injury occurs during H2O2 exposure. Activation of proteases may cause the reduction in catalase activity in this setting.