The drug-sensitivity and cross-resistance profiles of cloned cell lines of Adriamycin-sensitive and -resistant P388 murine leukemia have been further characterized. A range of drug sensitivity that was more than 50,000-fold was observed for Adriamycin-sensitive cells; the most potent cytotoxic agent was the Adriamycin analog, 3'-(3-cyano-4-morpholinyl)-3'-deamino adriamycin, and the least active compound was vinblastine. Adriamycin-resistant cells, which express the multidrug resistance phenotype, were cross-resistant to the DNA topoisomerase II interactive drugs: actinomycin D, daunorubicin, mitoxantrone, etoposide, and 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, to the vinca alkaloids: vincristine and vinblastine, and to colchicine but not to the Adriamycin analog, 3'-(3-cyano-4-morpholinyl)-3'-deamino adriamycin or the alkylating agent, melphalan. These findings are consistent with other studies suggesting that 3'-(3-cyano-4-morpholinyl)-3'-deamino adriamycin acts as an alkylating agent. Studies with DNA topoisomerase II interactive agents, including mitoxantrone, the DNA intercalator, and etoposide, the epipodophyllotoxin, showed that, as with Adriamycin, cytotoxicity correlated closely with the formation of DNA double-strand breaks.