A tk+/- mouse embryonic stem (ES) cell line, designated 1G2, has been created in which one allele of the thymidine kinase (tk) gene was inactivated by targeted homologous recombination. This line is an analog of the mouse lymphoma tk+/- L5178Y cell line, which is used widely to assess the mutagenicity of chemical agents. Treatment of 1G2 cells with the alkylating agent N-ethyl-N-nitrosourea (ENU) resulted in a dose-related increase in trifluorothymidine-resistant colonies. Mutant frequencies of 152 and 296 per 10(6) cells were determined for 0.1 and 0.3 mg/ml doses of ENU, compared with a spontaneous mutant frequency of 15 per 10(6) cells. The data indicate that tk+/- 1G2 ES cells may be useful for the creation of a transgenic mouse model for assessing in vivo mutation using an endogenous autosomal gene.