The Ag receptor on B lymphocytes is a multimeric complex that is composed of an Ag-specific component, surface Ig, which is noncovalently associated with at least two other proteins, Igα and Igβ. These are the glycoprotein products of the B lineage-restricted mb-1 and B29 genes and are crucial for the cell surface expression and function of the Ag receptor on B lymphocytes. To better understand the regulation of mb-1, we have cloned and sequenced a 5.7-kb genomic DNA fragment that contained the human gene. The overall structure of human mb-1 is very similar to that of the murine gene, including the number and approximate size of exons. The promoter region lacks a TATA element, but contains two copies of an early B cell factor-binding motif, which previously has been shown to be important for murine mb-1 expression. Other structural features include two nuclear factor-κB binding sites at the 5' end of the gene and a long stretch of AG rich-sequence between exons 3 and 4, downstream of an Alu repeat sequence that contains a potential stem-loop structure. The mb-1 gene was localized to chromosome 19q13.2-13.3 by a combination of two methods, PCR amplification of DNA from a somatic cell hybrid-mapping panel and fluorescence in situ hybridization. An examination of the methylation pattern revealed a striking correlation between demethylation in the 5' region of the gene and expression of mb-1. The demethylated HpaII/MspI sites are adjacent to the nuclear factor-κB-binding motifs, which suggests a role for this transcription factor in the regulation of human mb-1 gene expression.