Tk +/− transgenic mice were created using an embryonic stem cell line in which one allele of the endogenous thymidine kinase ( Tk) gene was inactivated by targeted homologous recombination. Breeding Tk +/− parents produced viable Tk −/− knockout (KO) mice. Splenic lymphocytes from KO mice were used in reconstruction experiments for determining the conditions necessary for recovering Tk somatic cell mutants from Tk +/− mice. The cloning efficiency of KO lymphocytes was not affected by the toxic thymidine analogues 5-bromo-2′-deoxyuridine (BrdUrd) or trifluorothymidine (TFT), or by BrdUrd in the presence of lymphocytes from Tk +/− animals; however, it was easier to identify clones resistant to BrdUrd than to TFT when Tk +/− cells were present. Tk +/− mice were treated with vehicle or 100 mg/kg of N-ethyl- N-nitrosourea (ENU), and after 4 months, the frequency of Tk mutant lymphocytes was measured by resistance to BrdUrd. The frequency of Tk mutants was 22±5.9×10 −6 in control animals and 80±31×10 −6 in treated mice. In comparison, the frequency of Hprt mutant lymphocytes, as measured by resistance to 6-thioguanine, was 2.0±1.2×10 −6 in control animals and 84±28×10 −6 in the ENU-treated mice. Analysis of BrdUrd-resistant lymphocyte clones derived from the ENU-treated animals revealed point mutations in the non-targeted Tk allele. These results indicate that the selection of BrdUrd-resistant lymphocytes from Tk +/− mice may be used for assessing in vivo mutation in an endogenous, autosomal gene.