A genomic DNA clone for 1 alpha,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) 24-hydroxylase was isolated from a human chromosome 20 library. It spans 2.42 kb, containing the first two exons, the first and part of the second introns, and a 1.26 kb 5'-flanking region. Putative transcription cis-elements were revealed throughout the 5'-flanking region, including TATA box, CAAT box, GC boxes, vitamin D-responsive elements (VDRE), AP1, and AP2 sites. In a CAT reporter gene expression assay, the 24-hydroxylase promoter with its 1.2 kb 5'-flanking sequence elicits a 1,25-(OH)2D3-induced transactivation activity. Gel mobility shift assays of those putative DREs have identified that two different elements can form specific complexes with porcine intestinal nuclear extract (PINE). The specificity of VDRE-PINE complexes was verified by supershift assay with VDR-specific monoclonal antibody VXIE10B6. The proximal element VDREp (-172/-143) consists of three direct repeat half-sites, GAGTCAgcgAGGTGAgcgAGGGCG, in anti-sense orientation. The distal element VDREd (-293/-273) consists of two direct repeat half-sites, GCGTTCaccGGGTGT, also in anti-sense orientation. Both VDREs can direct a reporter gene expression using a heterologous herpes simplex virus thymidine kinase (TK) promoter in a 1,25-(OH)2D3-dependent fashion. Further characterization of these VDREs in various constructs with either a native or TK promoter suggests that both VDREs are required for the optimal induction of 24-hydroxylase expression by 1,25-(OH)2D3.