Tumors derived from individuals with hereditary nonpolyposis colorectal cancer syndrome frequently demonstrate mutations in both alleles of hMSH2, a key gene in DNA mismatch repair (MMR). Sporadic tumors also frequently exhibit MMR deficiency. In keeping with the role of MMR in the maintenance of genome integrity, mice deficient in MSH2 via gene targeting demonstrate a high incidence of thymic lymphomas and small intestinal adenocarcinomas. To investigate the effects of MSH2 deficiency in normal tissues, mice containing a retrievable transgenic lacI reporter gene for mutation detection were crossed with MSH2-/- mice. Mice homozygous for MSH2 deficiency revealed 4.8, 11.0 and 15.2-fold elevations in spontaneous mutation frequency in DNA obtained from brain, small intestine, and thymus, respectively, as compared to heterozygous or wild-type mice. Mutations most frequently recovered from MSH2-/- mice were single base substitutions (77%), particularly base transitions (64%). Frameshifts occurred less frequently (19%) and fell within very short (3-5 bp) mononucleotide runs. Thus the number of key growth control genes potentially impacted by MMR deficiency extends beyond those containing repetitive sequences. These results highlight the capacity for MSH2 deficiency to serve as a potent driving force during the multi-step evolution of tumors.