The DNA mismatch repair gene hMLH1 is reported to function in mutation avoidance, cell cycle checkpoint control, the cytotoxicity of various DNA-damaging agents, and transcription-coupled nucleotide excision repair. Formal proof of the involvement of hMLH1 in these processes requires single gene complementation. We have stably expressed hMLH1 from a transfected cDNA in Mlh1-deficient mouse embryonic fibroblasts. Expression of hMLH1 restored normal levels of mPMS2 protein, reduced spontaneous base substitution and microsatellite mutations, increased sensitivity to the toxic effects of 6-thioguanine (6-TG), and restored 6-TG-induced cell cycle arrest. Our studies confirm that hMLH1 has an essential role in the maintenance of genomic stability and the potentiation of 6-TG cytotoxicity and provide a system for detailed structure/function analysis of the hMLH1 protein.