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 (ERRE, -241 bp), TTF-1/Nkx2.1 (TBE, -171 bp), USF1/2 (E-box, -80 bp), and Sp1 (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 bp 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.