The human vitamin D-binding protein (hDBP) gene exists in a cluster of four liver-expressed genes. A minimal hDBP transgene, containing a defined set of liver-specific DNase I hypersensitive sites (HSs), is robustly expressed in mouse liver in a copy-number-dependent manner. Here we evaluate these HSs for function. Deletion of HSI, located 5' to the promoter (kb -2.1) had no significant effect on hDBP expression. In contrast, deletion of HSIV and HSV from intron 1 repressed hDBP expression and eliminated copy number dependency without a loss of liver specificity. Chromatin immunoprecipitation analysis revealed peaks of histone H3 and H4 acetylation coincident with HSIV in the intact hDBP locus. This region contains a conserved array of binding sites for the liver-enriched transcription factor C/EBP. In vitro studies revealed selective binding of C/EBPalpha to HSIV. In vivo occupancy of C/EBPalpha at HSIV was demonstrated in hepatic chromatin, and depletion of C/EBPalpha in a hepatic cell line decreased hDBP expression. A nonredundant role for C/EBPalpha was confirmed in vivo by demonstrating a reduction of hDBP expression in C/EBPalpha-null mice. Parallel studies revealed in vivo occupancy of the liver-enriched factor HNF1alpha at HSIII (at kb 0.13) within the hDBP promoter. These data demonstrate a critical role for elements within intron 1 in the establishment of an autonomous and productive hDBP chromatin locus and suggest that this function is dependent upon C/EBPalpha. Cooperative interactions between these intronic complexes and liver-restricted complexes within the target promoter are likely to underlie the consistency and liver specificity of the hDBP activation.