Adriamycin (ADM, or doxorubicin hydrochloride) is an effective antineoplastic agent whose use is restricted by its well-described, dose-dependent cardiotoxicity. This study measures ADM DNA adduct formation by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thinlayer chromatography (2D-TLC), and quantitating the adducts with autoradiography and densitometry. Thirty-six male Sprague-Dawley rats are randomized to receive ADM at varying intraperitoneal (i.p.) injection concentrations: 0.9% saline i.p. controls, 4 mg/kg ADM i.p., and 6 mg/kg ADM i.p. Myocardial and pulmonary tissues are harvested 48 hours after i.p. injection for autoradiographic and histopathologic analyses. The results indicate differences in the amount and type of adduct formation as a function of ADM concentration. Increased partial depurination of dGMP and dAMP occurs with increasing ADM concentration at equal incubation times. This depurination correlates with the emergence of new adducts HM-dUMP, 8-OH-dGMP, HM-dCMP, and Me-dCMP. The quantification of these adducts can potentially represent an early marker of ADM cardiotoxicity and thereby optimize the efficacy of individual chemotherapy regimens while minimizing adverse effects.