Adriamycin is an anthracycline antineoplastic agent whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. A current hypothesis for adriamycin cardiotoxicity involves free radical oxidative stress. To investigate this hypothesis in a model system, we applied the technique of immunochemical detection of protein carbonyls, known to be increased in oxidized proteins, to study the effect of adriamycin on rat erythrocyte membranes. Erythrocytes obtained from adriamycin-treated rats demonstrated an increase of carbonyl formation in their membrane proteins. Yet, in separate experiments when adriamycin was incubated with rat erythrocyte ghosts, there was no significant increase of membrane protein carbonyls detected. In contrast, isolated erythrocytes incubated with an adriamycin-Fe3+ complex exhibited a robust carbonyl incorporation into their membrane proteins in a time-dependent manner. The level of carbonyl formation was dependent upon the concentration of Fe3+ known to form the adriamycin-Fe3+ complex. When the time course between protein carbonyl formation and lipid peroxidation was compared, protein carbonyl detection occurred earlier than lipid peroxidation as assayed by thiobarbituric acid reactive substances formation. These results are consistent with the notion that oxidative modification of membrane proteins may contribute to the development of the acute adriamycin-mediated toxicity.