Paramutation is an interaction between alleles that leads to a heritable change in the expression of one allele. In B'/B-I plants, B-I (high transcription) always changes to B' (low transcription). The new B' allele retains the low expression state in the next generation and paramutates B-I at a frequency of 100%. Comparisons of the structure and expression of B' with that of a closely related allele that does not participate in paramutation demonstrated that transcription from the same promoter-proximal sequences is not sufficient for paramutation. Fine-structure recombination mapping localized sequences required for B' expression and paramutation. The entire 110 kb upstream of the B' transcription start site was cloned and sequenced and the recombination breakpoints were determined for 12 recombinant alleles. Sequences required for expression and paramutation mapped to distinct regions, 8.5-49 kb and 93-106 kb upstream of the B' transcription start site, respectively. Sequencing and DNA blot analyses indicate that the B' region required for paramutation is mostly unique or low copy in the maize genome. These results represent the first example of long-distance regulatory elements in plants and demonstrate that paramutation is mediated by long-distance cis and trans interactions.