The vitamin D receptor (VDR) regulates steroid and drug metabolism by inducing the genes encoding phase I and phase II enzymes. SULT2A1 is a liver- and intestine-expressed sulfo-conjugating enzyme that converts the alcohol-OH of neutral steroids, bile acids and drugs to water-soluble sulfated metabolites. 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] induces SULT2A1 gene transcription following the recruitment of VDR to the vitamin D responsive chromatin region of SULT2A1. A composite element in human SULT2A1 directs the 1,25-(OH)₂D₃-mediated induction of natural and heterologous promoters. This element combines a VDR/RXR--binding site (VDRE), which is an imperfect inverted repeat (IR2) of AGCTCA, and a C/EBP-binding site located 9 bases downstream to VDRE. The binding sites were identified by EMSA, antibody supershift and DNAse 1 footprinting. C/EBP- at the composite element plays an essential role in the VDR regulation of SULT2A1, since a) induction was lost for promoters with inactivating mutations at the VDRE or C/EBP element; b) SULT2A1 induction by 1,25-(OH)₂D₃ in C/EBP- deficient cells required the expression of cotransfected C/EBP-; c) C/EBP- did not substitute for C/EBP- in this regulation. VDR and C/EBP- were recruited concurrently to the composite element along with the coactivators p300, SRC-1 and SRC-2, but not SRC-3. VDR and C/EBP- associated endogenously as a DNA-dependent, co-immunoprecipitable complex, which was detected at a markedly higher level in 1,25-(OH)₂D₃-treated cells. These results provide the first example of the essential role of the interaction in cis between C/EBP- and VDR in directing 1,25-(OH)₂D₃-induced expression of a VDR target gene.