Microsatellites are important tools for plant breeding, genetics, and evolution, but few studies have analyzed their mutation pattern in plants. In this study, we estimated the mutation rate for 142 microsatellite loci in maize (Zea mays subsp. mays) in two different experiments of mutation accumulation. The mutation rate per generation was estimated to be 7.7 x 10(-4) for microsatellites with dinucleotide repeat motifs, with a 95% confidence interval from 5.2 x 10(-4) to 1.1 x 10(-3). For microsatellites with repeat motifs of more than 2 bp in length, no mutations were detected; so we could only estimate the upper 95% confidence limit of 5.1 x 10(-5) for the mutation rate. For dinucleotide repeat microsatellites, we also determined that the variance of change in the number of repeats (sigma(m)2) is 3.2. We sequenced 55 of the 73 observed mutations, and all mutations proved to be changes in the number of repeats in the microsatellite or in mononucleotide tracts flanking the microsatellite. There is a higher probability to mutate to an allele of larger size. There is heterogeneity in the mutation rate among dinucleotide microsatellites and a positive correlation between the number of repeats in the progenitor allele and the mutation rate. The microsatellite-based estimate of the effective population size of maize is more than an order of magnitude less than previously reported values based on nucleotide sequence variation.