A non-P-glycoprotein-mediated mechanism of multidrug resistance (non-Pgp MDR) has been identified in doxorubicin-selected sublines of the human non-small cell lung carcinoma cell line SW-1573. These sublines are cross-resistant to daunorubicin, VP16-213, Vinca alkaloids, colchicine, gramicidin D, and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). They accumulate less drug than the parental cells and their resistance is not due to the MDR1-encoded P-glycoprotein, as the resistant cell lines have lost the low amount of MDR1 mRNA detectable in parental cells. Here we show that the resistant cell lines also contain less topoisomerase II mRNA and enzyme activity than the parental cells. This might contribute to the resistance of these lines to drugs interacting with topoisomerase II, such as doxorubicin, daunorubicin, and VP16-213, but cannot account for the resistance to the other drugs. We have tested whether all properties of the non-Pgp MDR cell lines cosegregate in somatic cell fusions between lethally gamma-irradiated, resistant donor cells and drug-sensitive acceptor cells. Whereas a MDR phenotype with reduced drug accumulation and the loss of MDR1 P-glycoprotein mRNA were cotransferred to the acceptor cells, the decrease in topoisomerase II gene expression was not. We conclude that the MDR phenotype, the reduced drug accumulation, and the loss of MDR1 P-glycoprotein mRNA are genetically linked. They might be due to a single dominant mutation, which does not cause the alteration in topoisomerase II.