Doxorubicin (Adriamycin) is one of the most commonly used chemotherapeutic drugs and exhibits a wide spectrum of activity against solid tumors, lymphomas, and leukemias. Doxorubicin is classified as a topoisomerase II poison, although other mechanisms of action have been characterized. Here, we show that doxorubicin-DNA adducts (formed by the coadministration of doxorubicin with non-toxic doses of formaldehyde-releasing prodrugs) induce a more cytotoxic response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic topoisomerase II-mediated cellular responses (as observed by employing topoisomerase II catalytic inhibitors and HL-60/MX2 cells). Apoptosis induced by doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overexpressed Bcl-2, suggesting that adducts induce a classic mode of apoptosis. A reduction in the level of topoisomerase II-mediated double-strand-breaks was also observed with increasing levels of doxorubicin-DNA adducts and increased levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic topoisomerase II poison mode of cell death by doxorubicin alone. Collectively, these results indicate that the presence of formaldehyde transfers doxorubicin from topoisomerase II-mediated cellular damage to the formation of doxorubicin-DNA adducts, and that these adducts are more cytotoxic than topoisomerase II-mediated lesions. These results also show that doxorubicin can induce apoptosis by a non-topoisomerase II-dependent mechanism, and this provides exciting new prospects for enhancing the clinical use of this agent and for the development of new derivatives and new tumor-targeted therapies.