Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is inreased following acute lung injury. Given the imporance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoorms by lung epithelial cells, we hypothesized that conerved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expresion by C/EBPs. Five SP-D motifs (-432, -340, -319, -140, and -90) homologous to the C/EBP consensus seuence specifically bound to C/EBPs in gel shift assays, and four of the five sites (-432, -340, -319, and -90) efficiently competed for the binding of C/EBPα,C/EBPβ, C/EBPPσ, to consensus oligomers. Cotransfection of C/EBPαC/EBPβ, or C/EBPσ, cDNA in H441 lung adenoarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containng 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences uptream of -205. Independent site-directed mutagenesis of the sites at -432, -340, and -319 reduced C/EBP mediated activation by sigma;50% and mutagenesis of the site at -432 in combination with either of the tandem sites at -340 and -319 blocked activation. The conserved AP-1 element at -109 was required for maximal prooter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter acivity of SP-D.