The Pms2 gene has been implicated in hereditary colon cancer and is one of several mammalian homologs of the Escherichia coli mutL DNA mismatch repair gene. To determine the effect of Pms2 inactivation on genomic integrity in vivo, hybrid transgenic mice were constructed that carry targeted disruptions at the Pms2 loci along with a chromosomally integrated mutation reporter gene. In the absence of any mutagenic treatment, mice nullizygous for Pms2 showed a 100-fold elevation in mutation frequency in all tissues examined compared with both wild-type and heterozygous litter mates. The mutation pattern in the nullizygotes was notable for frequent 1-bp deletions and insertions within mononucleotide repeat sequences, consistent with an essential role for PMS2 in the repair of replication slippage errors. Further, the results demonstrate that high rates of mutagenesis in multiple tissues are compatible with normal development and life and are not necessarily associated with accelerated aging. Also, the finding of genetic instability in all tissues tested contrasts with the limited tissue distribution of cancers in the animals, raising important questions regarding the role of mutagenesis in carcinogenesis.