We have isolated a Chinese hamster lung cell line resistant to 9-hydroxyellipticine (DC-3F/9-OH-E) which is also cross-resistant to topoisomerase II inhibitors such as amsacrine and etoposide. In this work we have studied quantitatively both DNA topoisomerase II activity by decatenation of kinetoplast DNA and drug-stimulated DNA cleavage of pBR 322. DNA topoisomerase II activity of DC-3F/9-OH-E nuclear extract was reduced by 3.5-fold as compared to that from DC-3F (sensitive parent) nuclear extract. We also found that DC-3F/9-OH-E nuclear extracts have a reduced capacity to induce in vitro topoisomerase II-mediated DNA cleavage upon stimulation by etoposide and amsacrine (7- and 10-fold respectively). Besides, mixing nuclear extracts from both sensitive and resistant cells indicates that either the enzyme in resistant cells is modified or a modulating factor is associated to it. Our results suggest that the resistance of the DC-3F/9-OH-E cell line to topoisomerase II inhibitors might be due to both a reduced amount of the enzyme and its reduced ability to form the cleavable complex in the presence of drugs.