Analysis of variation at microsatellite DNA loci is widely used in studies of parentage, linkage and evolutionary history. The utility of microsatellites is primarily due to high levels of allelic diversity, believed to reflect mutation rates orders of magnitude higher than base pair substitutions at single-copy genes. For humans, mice, rats and pigs, microsatellite mutation rates have been estimated at 10(-3)-10(-5). However, a recent study comparing microsatellite variation in humans with non-human primates suggests that microsatellite mutation rates may vary considerably across taxa. We measured mutation rates of 24 microsatellite loci in mutation accumulation lines of Drosophila melanogaster. Surprisingly, only a single mutation was detected after screening 157,680 allele-generations, yielding an estimated average mutation rate per locus of 6.3 x 10(-6), a mutation rate considerably lower than reported for various mammals. We propose that the comparatively low mutation rate is primarily a function of short microsatellite repeat lengths in the D. melanogaster genome.