We have previously shown that the multidrug-resistant EHR2/DNR+ cells, which overexpress P-glycoprotein, accumulate only about 20-30% of daunorubicin at steady state compared to the sensitive cells. These cells have been thought to be a "pure" P-glycoprotein cell line. We now report that the EHR2/DNR+ cells exhibit decreased DNA topoisomerase II catalytic activity. We also found that the amount of immunoreactive DNA topoisomerase II from these cells is about one-third that seen in the drug-sensitive cell line. In agreement with the decreased activity and amount of topoisomerase II, the number of DNA-protein complexes stabilized by teniposide (VM-26) was reduced by about 50% in nuclear extracts from EHR2/DNR+ cells. Furthermore, using an intact cell assay for DNA protein complexes, we found that the VM-26-stimulated complexes formed in the drug-resistant cells never reached the level seen in the drug-sensitive cells. Verapamil and Cremophor EL block P-glycoprotein-mediated efflux of "natural product" drugs and increase their accumulation in resistant cells. Coincubation of the EHR2/DNR+ cells with VM-26 and either of these modulators increased the number of complexes formed in the resistant cells. However, neither modulator increased the number of topoisomerase II-DNA complexes in the drug-resistant cells to the level seen in the EHR2 cells. We conclude that the resistance of EHR2/DNR+ cells is due in part to reduced amounts of DNA topoisomerase II. Furthermore, we note that a single cell line can express features of both P-glycoprotein-associated multidrug resistance and altered topoisomerase II-associated multidrug resistance.