In a recent study, we reported that the combined average mutation rate of ten di-, six tri-, and eight tetranucleotide repeats in Drosophila melanogaster was 6.3x10-6 mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10-?2 to 10-5 per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the same mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3x10-6 per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation and find that tri- and tetranucleotide repeats mutate at a rate 6.4 and 8.4 times slower than dinucleotide repeats, respectively. The slower mutation rate of tri- and tetranucleotide repeats appears to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit length of microsatellite decreases.