The mouse surfactant protein B (SP-B) gene was isolated from a DBA/2J EMBL3 genomic library, and the nucleotide sequence and transcriptional start site was determined. The SP-B gene was comprised of 11 exons and 10 introns and spanned 9,882 bases. Repetitive elements of the B1, B2, and R families and several other motifs were located within nontranslated regions of the gene. The 5' flanking region of the gene was utilized to express a bacterial chloramphenicol acetyl transferase reporter gene in mouse and human pulmonary adenocarcinoma cell lines. Transcriptionally active regions of the promoter were identified that determined lung epithelial cell-specific gene expression. Regions of the gene promoter shared between the mouse and human SP-B gene were used to locate putative transcription factor binding sites for thyroid transcription factor-1 and hepatocyte nuclear factor-3. The 5' flanking region of the murine SP-B contains cell-specific cis-active elements that regulate SP-B expression in the respiratory epithelium.