Secondary alcohol metabolites have been proposed to mediate chronic cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines. In this study, NADPH-supplemented human cardiac cytosol was found to reduce the carbonyl group in the side chain of the tetracyclic ring of DOX, producing the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in the level of alcohol metabolite formation was observed by replacing DOX with epirubicin (EPI), a less cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the hydroxyl group at C-4 in the amino sugar bound to the tetracyclic ring (daunosamine). A similar decrease was observed by replacing DOX with MEN 10755, a novel anthracycline with preclinical evidence of reduced cardiotoxicity. MEN 10755 is characterized by the lack of a methoxy group at C-4 in the tetracyclic ring and by intercalation of 2, 6-dideoxy-L-fucose between daunosamine and the aglycone. Multiple comparisons with methoxy- or 4-demethoxyaglycones, and a number of mono- or disaccharide 4-demethoxyanthracyclines, showed that both the lack of the methoxy group and the presence of a disaccharide moiety limited alcohol metabolite formation by MEN 10755. Studies with enzymatically generated or purified anthracycline secondary alcohols also showed that the presence of a disaccharide moiety, but not the lack of a methoxy group, made the metabolite of MEN 10755 less reactive with the [4Fe-4S] cluster of cytoplasmic aconitase, as evidenced by its limited reoxidation to the parent carbonyl anthracycline and by a reduced level of delocalization of Fe(II) from the cluster. Collectively, these studies (i) characterize the different influence of methoxy and sugar substituents on the formation and [4Fe-4S] reactivity of anthracycline secondary alcohols, (ii) lend support to the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they demonstrate that the less cardiotoxic EPI and MEN 10755 share a reduction in the level of formation of such metabolites, and (iii) suggest that the cardiotoxicity of MEN 10755 might be further decreased by the reduced [4Fe-4S] reactivity of its alcohol metabolite.