We have isolated and characterized murine surfactant protein (SP-B) cDNAs and determined both the cellular distribution and developmental expression of SP-B mRNA. The nucleotide sequence of the composite SP-B cDNAs and cloned genomic SP-B DNA predicted a primary translation product of 377 amino acids (molecular mass = 41.7 kDa) that contained a single asparagine linked glycosylation site. The 410 base 3' untranslated region contained a novel sequence consisting of a (CCA)21 repeat. Tissue specificity and developmental expression of SP-B mRNA was assessed by Northern blot analysis. A single 1.6-kb mRNA species was detected specifically in lung tissue. In fetal mouse lung SP-B mRNA was detected at low levels on day 15 of gestation and increased markedly between days 16 and 17, reaching adult levels by birth. In situ hybridization analysis of SP-B mRNA demonstrated SP-B expression primarily in bronchiolar and alveolar epithelium, although discrete bronchial cells also contained the SP-B mRNA. The murine SP-B mRNA was expressed in proximal and distal epithelial cells within the respiratory tract and shares a close structural relationship to SP-B from other species.