In order to investigate the oxidative component of adriamycin-induced cardiotoxicity in the rat, we used neonatal cardiac myocytes in culture. All incubations, with or without adriamycin (ADM), were performed under normoxic circumstances and additionally under circumstances which make cells more vulnerable towards oxidative challenges: hyperoxia or treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). ADM (100 microM) produced a decrease in the beating rate and enzyme release of the cultures. These effects were potentiated by hyperoxia and by BCNU treatment. Cellular GSH was depleted due to ADM. However, no significant increase in GSSG could be detected, even if the O2-concentration was increased. Lipid peroxidation, measured as thiobarbituric acid reactive material, could be detected only in case ADM plus additional stress were given to the cells. It is concluded that redox-cycling of ADM occurs in rat cardiac myocytes. Formation of ADM-glutathione conjugates or mixed disulfides is strongly indicated. From this it can be inferred that ADM-toxicity in cardiac cells may involve an oxidative mechanism. An important role for the glutathione system is indicated in the detoxification of reactive intermediates. In addition the results implicate that neonatal rat heart cell cultures provide a good screening system for the evaluation of oxidative challenges in the cardiotoxic action of anthracycline analogs.