Microsatellites are simple, tandem DNA repeats that represent unstable regions of the genome. They undergo frequent changes in tract length by base additions or deletions due to DNA polymerase slippage during replication. To characterize factors affecting the frequency of spontaneous mutations occurring in microsatellites in plants, a reporter system was used in Arabidopsis thaliana and tomato (Lycopersicon esculentum). The beta-glucuronidase (GUS) reporter system was used to measure the mutation frequency in various microsatellites (G(7), G(10), G(13), G(16), and C(16)) in somatic tissues. Our results indicate that this frequency increases with the number of repeats: a G(16) tract was almost 80-fold more mutable than a G(7) tract. Furthermore, the frequency of mutations depends on repeat orientation, as G(16) was 3-fold more mutable than C(16). The mutation rate was also found to differ markedly in Arabidopsis and tomato for an identical microsatellite. Indeed, Arabidopsis showed a 5-fold higher mutation frequency than tomato with the same G(7) reporter construct. Finally, mutation in a G(16) tract was frequent enough that mutations transmitted germinally to the next generation could be detected at a relatively high frequency.