Mutations in the human DNA mismatch repair (MMR) gene MLH1 are associated with hereditary nonpolyposis colorectal cancer (Lynch syndrome, HNPCC) and a significant proportion of sporadic colorectal cancer. The inactivation of MLH1 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of DNA damaging agents. To study the effect of MLH1 missense mutations on cancer susceptibility, we generated a mouse line carrying the recurrent Mlh1^G67R mutation that is located in one of the ATP-binding domains of Mlh1. Although the Mlh1^G67R mutation resulted in DNA repair deficiency in homozygous mutant mice, it did not affect the MMR-mediated cellular response to DNA damage, including the apoptotic response of epithelial cells in the intestinal mucosa to cisplatin, which was defective in Mlh1^−/− mice but remained normal in Mlh1^G67R/G67R mice. Similar to Mlh1^−/− mice, Mlh1^G67R/G67R mutant mice displayed a strong cancer predisposition phenotype. However, in contrast to Mlh1^−/− mice, Mlh1^G67R/G67R mutant mice developed significantly fewer intestinal tumors, indicating that Mlh1 missense mutations can affect MMR tumor suppressor functions in a tissue-specific manner. In addition, Mlh1^G67R/G67R mice were sterile because of the inability of the mutant Mlh1^G67R protein to interact with meiotic chromosomes at pachynema, demonstrating that the ATPase activity of Mlh1 is essential for fertility in mammals.