N-(2-Chloroethyl)-N-nitrosoureidodaunorubicin (AD 312), a novel semisynthetic compound with combined anthracycline and nitrosourea alkylating functionalities, circumvents resistance conferred by either reduced DNA topoisomerase II (topo II) or increased P-glycoprotein expression with less myelosuppression and cardiotoxicity than adriamycin (doxorubicin; ADR). Cellular resistance to AD 312 could arise from a novel mechanism that confers resistance to both functions simultaneously, or one or more mechanisms common to anthracyclines and/or alkylating agents. The mechanism contributing to AD 312 resistance was investigated following selection of AD 312-resistant murine J774.2 macrophage-like cells and human NCI-H460 non-small-cell lung carcinoma cells. Murine J/312-400 (> 4.7-fold) and human H/312-40 cells (6.3-fold) were cross-resistant to topo II inhibitors (ADR, teniposide, etoposide) and nitrosoureas (carmustine, lomustine) but remained sensitive to vinblastine, colchicine, and camptothecin. There was approximately a twofold decrease in topo II decatenation activity and protein. Decreased net intracellular drug accumulation was not observed. There were no increases in glutathione content or glutathione-S-transferase activity. Increased O6-methylguanine-DNA methyltransferase (MGMT) activity (2.3-fold) was detected in J/312-400, and AD 312 resistance was partially reversed by O6-benzylguanine, a potent inhibitor of MGMT activity. The results suggest that AD 312 resistance arose through selective pressure by both cytotoxic functions in a serial manner.