Surfactant protein B (SP-B) is essential for physiological function of pulmonary surfactant. In the present investigation, we isolated rabbit SP-B gene and determined its nucleotide sequence, transcription start site, and exon/intron organization. The coding region of rabbit SP-B gene is comprised of 6.8 kb of sequence and is organized into 10 introns and 11 exons. By deletion analysis we determined that a region of SP-B gene extending from -236 to +39 nucleotides is sufficient for high-level expression of CAT reporter gene in a cell type-specific manner in the pulmonary adenocarcinoma cell line NCI-H441. Deletion of 5'-flanking sequence to -140 and -71 nucleotides significantly reduced SP-B promoter activity, suggesting that the -236 to +39 region contains cis-DNA elements required for cell type-specific expression. The proximal promoter region of SP-B gene contained DNA sequence motifs for binding thyroid transcription factor 1 (TTF-1) (-113 to -97) and hepatocyte nuclear factor 3(HNF-3) (-91 to -81). Although SP-B gene sequence -140 to +39 did not support high level expression of CAT gene in NCI-H441 cells, it was capable of activation by TTF-1 in HeLa cells, suggesting that the -236 to -140 sequence plays an important role in cell type-specific activation of SP-B promoter.