Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. In a previous transgenic mouse analysis, the expression pattern of the endogenous amelogenin gene was recapitulated by a reporter gene driven by a 2. 2-kilobase mouse amelogenin proximal promoter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogenin promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (-70/+52) containing a CCAAT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBPalpha up-regulated the promoter activity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBPalpha-mediated transactivation and basal promoter activity. Electrophoresis mobility shift assays demonstrated that C/EBPalpha bound to its cognate site in the amelogenin promoter and that the binding was specific. Endogenous C/EBPalpha was detected in LS8 cells, and overexpression of exogenous C/EBPalpha in LS8 cells was able to increase the expression level of the endogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, ranging from 20 to 30% of the activity in ameloblast-like cells. Transient transfection experiments showed that C/EBPalpha transactivated the mouse amelogenin reporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Taken together, these data indicate that C/EBPalpha is a bona fide transcriptional activator of the mouse amelogenin gene in a cell type-specific manner.