Adriamycin (ADM) is a broad-spectrum antineoplastic antibiotic used to treat cancer patients. However, the usefulness of this drug is presently limited by the development of a dose-dependent cardiotoxicity. A current hypothesis for the ADM-induced cardiotoxicity is the production of reactive oxygen radicals by the drug. We utilized the fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFH/DA), in which fluorescence appears if reactive oxygen species (ROS) are present, to investigate the ability of ADM to generate reactive oxygen species and the potential protective effect of antioxidants in a cultured cardiomyocyte model. All three of the antioxidants (alpha-phenyl-tert-butyl nitrone (PBN), trolox, and 5-aminosalicylic acid (5-ASA)) tested in our ADM-treated myocytes provided protection against the oxidative stress induced by the drug. These findings suggest that antioxidants modulate ADM-induced oxidative stress, and they are discussed in terms of a possible therapeutic strategy in the prevention of cardiotoxicity resulting from ADM administration.