We have used microsatellite sequences to evaluate the influence of the mismatch repair system on mutation bias in D. melanogaster. While mismatch-proficient cells have the highest mutation rate at (GT)(n) repeats, (AT)(n) repeats were the least stable ones in spel1(-/-) flies lacking functional mismatch repair. Furthermore, the mutation spectrum of long microsatellite alleles in spel1(-/-) was slightly upward biased, resulting in a gain of repeats, whereas wild-type flies have a strong downward bias. Interestingly, this mismatch repair-mediated downward mutation bias is reflected in the genome composition of D. melanogaster. When compared to other species, D. melanogaster has significantly shorter microsatellites. Our results suggest that the mismatch repair system may have an important role in shaping genome composition.