The Clara cell secretory protein (CCSP) gene is a cell-specific differentiation marker for the bronchiolar Clara cell. Previous studies suggest that CCAAT/enhancer binding protein (C/EBP)alpha is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBPdelta in the bronchiolar epithelium as well as lower levels of C/EBPalpha. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBPalpha and C/EBPdelta activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBPdelta exhibits an approximately 2-fold higher transactivation potential than does C/EBPalpha. DNase I footprint analyses revealed a footprint region located at -100 to -62 bp, corresponding to two C/EBP-binding sites. Mutation of either site resulted in abolished or strikingly reduced transactivation of the CCSP promoter by C/EBPalpha and C/EBPdelta, as well as impaired binding of both factors, indicating that the two C/EBP-binding sites form a compound response element. In electrophoretic mobility shift assays, it was shown that C/EBPalpha and C/EBPdelta can bind to both C/EBP sites, whereas in DNase I footprint analyses, the interaction of C/EBPalpha with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBPalpha and C/EBPdelta preferentially form heterodimers at both binding sites. Cotransfections with C/EBPalpha and C/EBPdelta together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBPalpha-C/EBPdelta heterodimers. Our findings demonstrate that C/EBPalpha and C/EBPdelta regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation-dependent expression of CCSP.