Using a replica microwell method, four Chinese hamster lines which exhibit hypersensitivity to the topoisomerase I inhibitor camptothecin, designated CM1, CM2, CM3 and CM6, have been isolated. Their sensitivity towards camptothecin varied from 3.5- to 8.2-fold with relative sensitivity as follows: CM2 < CM3 < CM6 < CM1. Genetic analysis of the CM mutants has established that CM1, CM3 and CM6 fail to complement each other and can each be assigned to the irs2 (XRCC8) complementation group. The mutant CM2 could not be definitively assigned to a complementation group because it presented a semi-dominant phenotype. In contrast to their sensitivity to camptothecin, the four CM mutants were less sensitive (1.1- to 2.2-fold) to the topoisomerase II inhibitors etoposide and adriamycin, although CM1, CM3 and CM6 were more sensitive (2.5- to 3. 8-fold) to streptonigrin (a free radical generator and a topoisomerase II inhibitor). All four mutant lines displayed an increased sensitivity to the bifunctional alkylating agent mitomycin C (2.4- to 5.1-fold). Surprisingly, given their assignment to the irs2 (XRCC8) complementation group, CM1, CM3 and CM6 displayed only a minor increase in sensitivity to ionizing radiation (1.6-fold or less). Similar sensitivity of these CM mutants was observed for the radiomimetic compound bleomycin (1.7-fold sensitive or less). This study indicates that XRCC8 mutants are isolated at high frequency from the parent line V79 and that phenotypic heterogeneity amongst the irs2 (XRCC8) complementation group is greater than previously encountered. Mutations in different regions of the XRCC8 gene may be responsible for the differing cellular phenotypes. Hamster XRCC8 mutants show phenotypic similarities to cultured cells from ataxia telangiectasia and Nijmegen break syndrome (NBS) patients and are likely to be defective in the same pathway in which the ATM (ataxia telangiectasia-mutated) and the NBS genes operate.