Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutLhomologues. Germline mutations in several members of these families, MSH2, MSH6, MLH1, and PMS2, but not MSH3, are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3, we generated a mouse with a null mutation in this gene. Cells from Msh3−/− mice are defective in repair of insertion/deletion mismatches but can repair base-base mismatches. Msh3−/− mice develop tumors at a late age. When the Msh3−/− and Msh6−/− mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2−/− or Mlh1−/− mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.