Surfactant protein A (SP-A) is a member of the collectin family of innate host defense molecules expressed primarily in respiratory epithelial cells of the lung. SP-A concentrations are influenced by both cell-specific and ubiquitous nuclear proteins that regulate SP-A gene transcription in a cell-selective and temporally regulated manner. In this work, a consensus GATA-binding site (GBS) was identified at positions -69 to -64 of the mouse SP-A gene. The transcriptional activity of wild-type SP-A reporter constructs in HeLa cells was increased 5-10-fold when cotransfected with a GATA-6 expression plasmid. Deletion of the GBS completely blocked transactivation by GATA-6. Transfection of a construct expressing GATA-6-engrailed fusion protein inhibited basal expression of the SP-A/chloramphenicol acetyltransferase construct in MLE-15 cells. Nuclear extract proteins from MLE-15 cells bound to the GBS in the mouse SP-A gene, and a supershifted band was detected with a GATA-6-specific antibody. Transactivation of the wild-type SP-A constructs by GATA-6 increased transcriptional activity 7-10-fold, whereas thyroid transcription factor-1 (TTF-1) increased the activity of these constructs 12-18-fold. The effects of cotransactivating with both GATA-6 and TTF-1 expression constructs were additive. However, mutation of the TTF-1-binding sites alone or in combination decreased GATA-6 transactivation. Likewise, mutation of the GBS blocked TTF-1 activation of the SP-A promoter. In situ hybridization demonstrated GATA-6 mRNA in the peripheral epithelial cells of fetal mouse lung, consistent with the sites of SP-A expression. GATA-6 is expressed in respiratory epithelial cells and binds to a cis-acting element in the SP-A gene promoter, activating the transcriptional activity of the gene.