The anticancer drug Adriamycin is widely used in cancer chemotherapy and is classified as a topoisomerase II inhibitor. However, in the presence of formaldehyde, Adriamycin also forms high levels of DNA adducts. In this study, a new series of butyric acid and formaldehyde-releasing drugs related to AN9 (pivaloyloxymethyl butyrate) was assessed for their ability to facilitate Adriamycin-DNA adduct formation in Adriamycin-sensitive and -resistant cell lines (HL60 and HL60/MX2; MES-SA and MES-SA/Dx5). Drugs that released two molar equivalents of formaldehyde per mole of prodrug were superior in their ability to enhance adduct formation compared to those that released one molar equivalent. Adduct formation (as assessed by binding of radiolabeled Adriamycin to genomic DNA) was always lower in the resistant cell lines compared to the sensitive cell lines. However, in growth inhibition experiments, prodrug combinations were able to overcome Adriamycin resistance to varying degrees, and the combination of Adriamycin with selected prodrugs that release two moles of formaldehyde totally overcame resistance in HL60/MX2 cells. These HL60-derived cells express altered levels of topoisomerase II and also express a mutant form of the enzyme. Combinations of Adriamycin with selected prodrugs that release one or two moles of formaldehyde partially overcame P-glycoprotein-mediated resistance in MES-SA/Dx5 cells. Formaldehyde-releasing prodrugs (as single agents) overcame both forms of resistance in the two resistant cell lines, demonstrating that they were not substrates of these resistance mechanisms. Collectively, these results suggest that changing the mechanism via which Adriamycin exerts its anticancer effect by dramatically increasing adduct levels (requiring coadministration of formaldehyde-releasing prodrugs) may be a useful means of cancer treatment, as well as for overcoming Adriamycin-induced resistance.