The cytotoxic activities and subcellular localizations of clinically used and synthetic analogues of the anthracycline family of chemotherapeutic agents were studied. The structures of the anthracycline derivatives affected their cytotoxicity and the time required for these compounds to exert cytotoxic effects on tumor cells. Fluorescent DNA intercalator displacement experiments demonstrated that there was no correlation between the DNA intercalation properties and the cytotoxicity of the studied anthracycline derivatives. Confocal microscopy experiments indicated that structural differences led to differences in subcellular localization. All studied anthracycline derivatives were observed in lysosomes, suggesting that this organelle, which is involved in several processes leading to malignancy, may contain previously unidentified molecular targets for these antitumor agents.