DNA topoisomerase-targeting antitumor drugs are potent inducers of protein-concealed strand breaks in mammalian cells and act by trapping DNA topoisomerases on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes. It has been proposed that the cleavable complex is an unusual form of DNA damage that elicits cellular responses analogous to those caused by DNA damaging agents. The relationship between topoisomerase-targeting drug-induced damage and radiation-induced damage has been investigated by analyzing the properties of DNA topoisomerases in mouse L5178Y lymphoma strains that are cross-sensitive to topoisomerase I-II inhibitors and to UV light or X-ray irradiation. The strains are LY-R, isolated from L5178Y cells on the basis of increased resistance to ionizing radiation, and strain LY-S, isolated from LY-R cells following a spontaneous increase in the sensitivity to ionizing radiation. LY-S cells, deficient in the rejoining of DNA double-strand breaks, show enhanced sensitivity to topoisomerase II-targeting inhibitors, whereas LY-R cells have an increased sensitivity to UV radiation and to the topoisomerase I inhibitor, camptothecin. The cellular availability of DNA topoisomerase I and II and the sensitivity of the enzymes to their specific inhibitors have been measured in the two related strains. In the LY-R strain, we found a 30% decrease in topoisomerase I content but no difference in camptothecin sensitivity, while no quantitative or qualitative differences were observed for the topoisomerase II. The results indicate that variations in sensitivity of the L5178Y strains to topoisomerase inhibitors are unlikely to be related to primary defects of the target enzymes, and thus it is possible that common pathways exist for processing of topoisomerase- and radiation-induced damage.