Surfactant protein A (SP-A) is expressed in lung alveolar type II cells and bronchiolar Clara cells. We have identified two active regions in the promoter of the rat SP-A gene by deletion analysis of a plasmid containing 163 bp before the start of transcription (163 bp), linked to a reporter gene. Constructs were transfected into lung cell lines derived from each of the cell types that produces SP-A. We found a novel region of promoter activity at ~90 bp before the transcriptional start (SP-A₉₀). Mutation of four nucleotides in SP-A₉₀ that are highly conserved among species (92 to 89 bp) decreased expression of the SP-A construct by ~50% in both cell lines. Electrophoretic mobility shift analysis showed specific binding to SP-A₉₀ by nuclear proteins from the cell lines, as well as from rat lung and liver. The electrophoretic mobility of the bands shifted by lung nuclear proteins changed late in fetal development. Although in the Clara cell line no reduction of promoter activity was seen on deletion of the region upstream of SP-A₉₀, in the type II cell line, deletion of residues 163 to 133 did reduce activity by ~50%. This region contains a recognition element for thyroid transcription factor-1 (TTF-1). Endogenous TTF-1 binding activity was substantially higher in the type II cell line than in the Clara cell line, but cotransfection of a TTF-1 expression plasmid enhanced expression of the SP-A construct better in the Clara cell line than in the type II cell line. These results suggest that the recognition element for TTF-1 has varying activity in the lung cell lines of different origin due to the availability of TTF-1.