Vitamin D is enzymatically modified to more than 35 metabolites. While many of these are thought to represent degradation products, some have been shown to exhibit biological activity. We tested whether 3-epi-1α,25-dihydroxyvitamin D 3 (3-epi-1α,25(OH) 2 D 3 ), 1α,25-dihydroxy-24-oxo-vitamin D 3 (1α,25(OH) 2 -24-oxo-D 3 ), and 1α,25(OH) 2 D 3 -26,23-lactone can stimulate transcription of vitamin D responsive genes. MC3T3-E1 cells transfected with a 25-hydroxyvitamin D 24-hydroxylase (CYP24) promoter construct displayed a 6 fold response when treated with either 1α,25(OH) 2 D 3 or 3-epi-1α,25(OH) 2 D 3 . Caco-2 cells were transfected with the wild type CYP24 promoter construct, or a Vitamin D Response Element (VDRE)-mutated form. Cells acquiring the wild type reporter responded to 1α,25(OH) 2 D 3 and 3-epi-1α,25(OH) 2 D 3 but not cells which acquired the mutated reporter. Additionally, VDR-negative COS-7 cells transfected with the wild type promoter responded (approximately 13 fold) to 1α,25(OH) 2 D 3 and 3-epi-1α,25(OH) 2 D 3 , only when co-transfected with the VDR. These results were confirmed using shorter incubation times and serum-free conditions. This strongly suggested that 3-epi-1α,25(OH) 2 D 3 mediates its effects through the VDR and its cognate binding site. Similar results were obtained with 1α,25(OH) 2 -24-oxo-D 3 using VDR-negative P19 cells. We could never detect activity from 1α,25(OH) 2 D 3 -26,23-lactone on vitamin D-responsive target promoters. Our results firmly conclude that both 3-epi-1α,25(OH) 2 D 3 and the 1α,25(OH) 2 -24-oxo-D 3 elicit their biological effects by acting through the VDR/VDRE.