The b locus of Zea mays encodes a transcriptional activator of the anthocyanin biosynthetic pathway. The B-Peru allele is expressed in the aleurone layer of the seed, which results in dark purple pigmentation of this tissue. An unstable Mutator-induced B-Peru mutant allele, b-Perum220, displays weak, variable pigment and a high germinal reversion rate not characteristic of other Mutator insertions. Characterization of relevant regions of b-Perum220 revealed a Mu2 element insertion in one copy of a 534 bp sequence. This 534 bp sequence is tandemly triplicated in the progenitor B-Peru allele, upstream of the B-Peru transcription start site. In addition to the Mu2 insertion, the b-Perum220 allele contains a newly formed large tandem duplication of 4.0 kb, which includes the promoter region and the first three exons of the B-Peru gene. The Mu2 element does not reside at any of the duplication breakpoints. The molecular study of eleven independent germinal revertants revealed five structural classes including structures in which the 4.0 kb tandem duplication is partially or completely deleted, the Mu2 element is partially or completely deleted, or a combination of these events has occurred. We hypothesize that most of the revertants arose by unequal recombination between the duplicated regions. Based on these structural analyses, models are discussed to explain the reduced b gene expression in b-Perum220.