Surfactant protein-A (SP-A) gene expression is developmentally regulated in fetal lung type II cells in concert with surfactant glycerophospholipid synthesis. In studies using transfected type II cells, we characterized a nuclear receptor element (NRE(SP-A), 5'-TGACCTTA-3') at -242 bp in the 5'-flanking sequence of human SP-A2 (hSP-A) gene that is essential for basal and cAMP-induced expression. NRE(SP-A) has high sequence similarity to the consensus binding site for estrogen-related receptor (ERR). In the present study, we observed that ERRalpha and ERRgamma, but not ERRbeta, were expressed in human fetal lung type II cells. In vitro transcribed/translated ERRalpha and ERRgamma bound to the NRE(SP-A); DNase I footprinting using bacterially expressed ERRalpha revealed a single DNase I protected region that included NRE(SP-A). In transient transfection assays of COS-7 and primary cultures of lung type II cells, ERRalpha acting through NRE(SP-A) increased hSP-A promoter activity, whereas ERRgamma had no effect. ERRalpha overexpression in lung type II cells enhanced cAMP induction of endogenous hSP-A expression, whereas cotransfection of protein kinase A catalytic subunit enhanced ERRalpha stimulation of hSP-A promoter activity in lung adenocarcinoma cells. Mice homozygous null for the ERRalpha gene manifested decreased SP-A expression relative to wild-type and heterozygous littermates. The ERRalpha-specific inverse agonist XCT790 inhibited cAMP induced hSP-A expression in human fetal lung type II cells in a concentration-dependent manner, suggesting a role of peroxisome proliferator-activated receptor-gamma coactivator 1alpha. These findings suggest that ERRalpha acting through NRE(SP-A) is an important mediator of hSP-A gene expression and its induction by cAMP.