A selectable system has been used to determine mutation rates within a microsatellite sequence in human cancer cell lines with or without defects in mismatch repair. A sequence consisting of 17 repeats of poly (dC-dA).poly(dT-dG) [abbreviated as (Ca)17] was inserted near the 5' end of the bacterial neomycin-resistance gene in a plasmid vector, such that the reading frame of the neo gene is disrupted. This plasmid was introduced into cancer cell lines, where it became integrated into the cellular genome. Clones with insertions or deletions of CA-repeats that restored the normal reading frame of the neo gene were selected in G418, and mutation rates were determined by fluctuation analysis. The rates of reversion in LoVo cells, which are deficient for hMSH2, were about one in a thousand per generation, which is approximately two orders of magnitude higher than in the repair-proficient HT-1080 human fibrosarcoma cell line. The mutation rates in H6 cells, which are derived from the hMLH1-deficient HCT116 line, were more heterogeneous than in LoVo, but all were considerably higher than in the repair-proficient line. Nearly all of the revertants of the repair-deficient lines had deletions of a single CA-repeat from the microsatellite sequence, whereas repair-proficient cells had a broader spectrum of mutations.