Expression of the human surfactant protein-A2 (hSP-A2) gene is lung specific, occurs in type II and Clara cells, and is developmentally and hormonally regulated in fetal lung. Using transfected human fetal type II cells, we previously observed that ∼300 bp of 5′-flanking DNA mediated cAMP and interleukin-1 (IL-1) stimulation and dexamethasone (Dex) inhibition of hSP-A2 promoter activity. This region contains response elements for estrogen-related receptor α element (ERRE, −241 bp), thyroid transcription factor (TTF)-1/Nkx2.1 (TTF-binding protein, −171 bp), upstream stimulatory factor 1/2 (E-box, −80 bp), and stimulatory protein (Sp) 1 (G/T-box, −62 bp), which are essential for basal and cAMP induction of hSP-A2 expression. To define genomic regions necessary for developmental, hormonal, and tissue-specific regulation of hSP-A2 expression in vivo, we analyzed transgenic mice carrying hGH reporter genes comprised of 313 bp of hSP-A2 gene 5′-flanking DNA ± mutation in the TBE or 175 bp of 5′-flanking DNA, containing TBE, E-box and G/T-box, but lacking ERRE. Transgenes containing 313 or 175 bp of hSP-A2 5′-flanking DNA were expressed in a lung cell-specific manner and developmentally regulated in concert with the endogenous mouse SP-A gene. In cultured lung explants from hSP-A₋₃₁₃:hGH transgenic fetal mice, cAMP and IL-1 induced and Dex inhibited transgene expression. However, the 175-bp hSP-A2 genomic region was insufficient to mediate hormonal regulation of hSP-A2 promoter activity. The finding that expression of the hSP-A_−313TBEmut:hGH transgene was essentially undetectable in fetal lung and was not hormonally regulated in transgenic fetal lung explants underscores the critical importance of the TBE in lung cell-specific, developmental, and hormonal regulation of hSP-A2 gene expression.