Dinucleotide repeats, because of their repetitive nature, are prone to frameshift mutations, most likely via a DNA-polymerase slippage mechanism. Mutation rates in microsatellite DNA sequences are high in mismatch repair-defective cells. In normal cells, only estimates of maximal rates of mutation in microsatellites have been possible previously, because of the low sensitivity of screening assays for mutations in endogenous sequences. We have measured the spontaneous mutation rate of a dinucleotide repeat in diploid human foreskin fibroblasts. In our system, the mutation target is a (CA)17 repeat contained within a stably integrated plasmid. The repeat disrupts the reading frame of a neomycin (neo) resistance gene within the plasmid. Cells containing frameshift mutations in the CA repeat that correct the reading frame of the neo gene are selected using the neo analogue G418. This system of measuring microsatellite mutation rates is highly sensitive, because there is a specific target within which mutations can be selected. Fluctuation analysis of cells containing the target DNA yielded mutation rates of <3.1 x 10(-8) to 44.8 x 10(-8) mutations/cell/generation. This is the first report of a direct measurement of a spontaneous mutation rate of a microsatellite sequence in normal human cells.