Two mutants of the Chinese hamster cell line V79–4 (irs1 and irs2) were previously isolated on the basis of their hypersensitivity (2- to 3-fold) to cell inactivation by ionizing radiation. One of these mutants, irs1, displays an unusual phenotype of cross-sensitivity to other varied genotoxic agents including UV light (2- to 3-fold), ethyl methanesulphonate (˜ 10-fold) and mitomycin C (˜ 60-fold). The possibility that these sensitivities might be due to more than one gene mutation in irs1 was investigated. Hybrids formed between irs1 and human lymphocytes were isolated in which the mitomycin C (MMC) sensitivity of irs1 was corrected by complementing human chromosomal material. These MMC-resistant hybrids and their subclones also showed concordant correction of the γ-ray, UV and EMS sensitivities of irs1, suggesting that a single gene defect is most likely responsible for the phenotype of irs1. In addition it was shown that the MMC-sensitivity of irs1 is complemented by four CHO cell mutants (UV20, UV41, UV-1 and irs1SF), which also display extreme sensitivity to MMC. Mutants irs1, irs1SF and UV-1 define three new complementation groups for MMC sensitivity. The biochemical nature of the ionizing radiation sensitivity of irs1 and irs2 was also investigated. The production and repair of DNA single- and double-strand breaks were studied using the techniques of alkaline and neutral elution, respectively. Irs1 and irs2 both showed repair kinetics for each lesion that are indistinguishable from wild-type. Analysis of the rate of DNA synthesis following γ-irradiation showed irs1 to have a dose-dependent inhibition similar to that of wild-type. In irs2, however, synthesis was significantly resistant to inhibition. The responses of irs1 and irs2 have some similarities to those of cells from persons with the cancer-prone syndrome ataxia telangiectasia (AT), but the dissimilarities between both irs mutants and AT cell lines suggest that the primary defects are distinct.