xrs-1 is an ionizing radiation sensitive Chinese hamster ovary (CHO) strain and has a defect in double strand break rejoining. It is also highly sensitive to topoisomerase II inhibiting anticancer drugs. Sensitivity is specific for those drugs that inhibit the breakage/reunion mechanism of topoisomerase II. xrs-1 and its parent strain CHO-K1 have equal levels of topoisomerase II activity, assayed by their ability to unknot complex knotted P4 head DNA. Drug stimulated protein-DNA complex formation was similar in xrs-1 and CHO-K1, showing that they accrued equal levels of drug induced lesions. Thus sensitivity most likely results from subsequent differences in the processing of these lesions rather than the rate of formation. Drug sensitivity is directly related to the xrs phenotype since drug and gamma-ray resistance are coordinately reactivated by azacytidine treatment. All six members of the xrs complementation group are hypersensitive to etoposide. Sensitivity is not a feature common to all X-ray sensitive mutants but is shown by another complementation group, which also has a defect in double strand break rejoining. These results thus demonstrate a correlation between an inability to rejoin double strand breaks and sensitivity to topoisomerase II targeting antitumor drugs.