The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D-3. Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides - 1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response. (c) 2007 Elsevier Inc. All rights reserved.