The hypervariable human minisatellite locus D7S22 (g3) is highly polymorphic. The allelic distribution in D7S22 features a size clustering of the alleles and a comparably low allelic diversity among small alleles. This reduced diversity could reflect a situation where some alleles are less likely to mutate than others. Several factors could explain such an effect, including allele size, variation in repeat composition, and allelic differences in nearby cis-acting elements affecting the mutation rate. We have characterized 40 de novo mutations found on Southern blots in a large amount of paternity-testing material. There is a significant excess of paternal mutations, and small size changes are most frequent. Mutation rate is affected by allele length, with highest rates in larger alleles. Alleles of the family groups with D7S22 mutations and 50 small alleles were analyzed by nucleotide sequencing. Two hundred thirty-six base pairs of the immediate flanking region upstream of the repeat array were PCR amplified and screened for point mutations by DNA sequencing of the PCR products. Two base substitution polymorphisms were identified: one C/G transversion and one A/G transition, 54 bp and 173 bp upstream of the repeat array, respectively. There is a significant association between mutation and occurrence of 54C, while association is not obvious between mutation rate and the 173A/G variants. There is a marked association between different flanking haplotypes and allele size, and within the smallest allele-size group, all alleles had the 54G/173A haplotype. Both allele size and allelic state at site 54 remain associated with mutation rate when the other factor is controlled. Possible mechanisms behind the variation in mutation rate in D7S22 are discussed.