We report on the isolation of the human BK B1 receptor gene from a human lung fibroblast genomic library. The gene is present as a single copy, spans more than 10 kilobases and includes three exons interrupted by two introns. The relative position of the exons was mapped using RNase protection assay, and genomic DNA and cDNA sequence matches. Exon and intron-exon boundaries, and 5', 3' flanking regions were sequenced. While the 5' untranslated region is distributed on all three exons, the coding region is located entirely on the third exon. The exon-intron junction sequences are highly conserved. Primer extension analysis mapped the transcriptional initiation site 21 base pairs downstream of a TATA sequence. Two Aluc sequences and two distinct functional promoters were found in the human BK B1 receptor gene.