1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ), the active metabolite of vitamin D, mediates many of its effects through the intranuclear vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of nuclear receptors. Vitamin D receptor can directly regulate gene expression by binding to vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1␣,25(OH) 2 D 3 have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the biologic activity of many naturally occuring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1␣,24R,25-trihydroxyvitamin D 3 (1␣,24R,25(OH) 3 D 3 ), 1␣,25-dihydroxy-3-epi-vitamin D 3 (1␣,25(OH) 2 -3-epi-D 3 ), 1␣,23S,25-trihydroxyvitamin D 3 (1␣,23S,25(OH) 3 D 3 ), and 1␣-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D 3 (1␣(OH)-24,25,26,27-tetranor-23-COOH-D 3 ; calcitroic acid) using the human G-361 melanoma cell line. Cells were cotransfected with a VDR expression plasmid and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1␣,25(OH) 2 D 3 24-hydroxylase (CYP24) promoter. Treatment with 1␣,25(OH) 2 D 3 or the metabolites 1␣,24R,25(OH) 3 D 3 , 1␣,25(OH) 2 -3-epi-D 3 , and 1␣,23S,25(OH) 3 D 3 resulted in transactivation of both constructs in a time-and dose-dependent manner, and a postitive regulatory effect was observed even for calcitroic acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24 mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent hormone. A ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [ 3 H]-labeled 1␣,25(OH) 2 D 3 from VDR than the parent hormone. Thus, we report that several natural metabolites of 1␣,25(OH) 2 D 3 retain significant biologic activity mediated through VDR despite their apparent low affinity for VDR.