Clone B is a CHO cell line that shows a moderate mutator phenotype as a consequence of a defect in mismatch recognition. To identify the classes of mutation that accumulate spontaneously in a functional gene, we isolated and sequenced 54 clone B spontaneous mutants at the adenine phosphoribosyltransferase gene. This spectrum was compared to 42 mutants collected in the parental cells. Rates of AT-->TA transversions and frameshifts were strikingly increased in clone B (almost eight- and sixfold, respectively). Minor increases were also observed for GC-->TA transversions and GC-->AT transition rates. Frameshifts occurred in repeated sequences, and a large proportion were losses of 2 bases occurring in dinucleotide runs of a type similar to microsatellite sequences. AT-->TA transversions clustered in regions of secondary structure and their formation might be explained by slippage-mediated mechanisms. These data indicate that an important function of mismatch recognition is in repair of extrahelical bases generated by misalignment during DNA replication.