High sulfur proteins are cysteine-rich proteins synthesized during the differentiation of hair matrix cells, and form hair fibers in association with hair keratin intermediate filaments. Rat high sulfur protein B2 genes were isolated after screening of a rat genomic library using the cDNA as a probe. Sequence analysis of a 4 kb fragment revealed two high sulfur protein genes, B2E and B2F. Both genes lacked introns, with B2F being located at 2 kb downstream of B2E. The 5' flanking regions of both genes had TATA and CAAT boxes, and consensus sequences of B2 genes. The upstream region of B2F had possible AP-1 and Sp-1 binding elements. The high sulfur protein B2E and B2F, which have putative 188 and 122 amino acids, respectively, comprised four distinct domains with a characteristic repetitive sequence. In situ hybridization indicated that the mRNA of high sulfur protein B2 was specifically localized in the cortex of the hair shaft, and northern blot analysis indicated that the expression of B2 increased in anagen and decreased in telogen, suggesting that high sulfur protein B2 synthesized in cortical cells during anagen contributes to the production of hair fibers.