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-1alpha,25-dihydroxyvitamin D(3) (3-epi-1alpha, 25(OH)(2)D(3)), 1alpha,25-dihydroxy-24-oxo-vitamin D(3) (1alpha, 25(OH)(2)-24-oxo-D(3)), and 1alpha,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 1alpha,25(OH)(2)D(3) or 3-epi-1alpha,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 1alpha,25(OH)(2)D(3) and 3-epi-1alpha,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 1alpha, 25(OH)(2)D(3) and 3-epi-1alpha,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-1alpha,25(OH)(2)D(3) mediates its effects through the VDR and its cognate binding site. Similar results were obtained with 1alpha,25(OH)(2)-24-oxo-D(3) using VDR-negative P19 cells. We could never detect activity from 1alpha,25(OH)(2)D(3)-26, 23-lactone on vitamin D-responsive target promoters. Our results firmly conclude that both 3-epi-1alpha,25(OH)(2)D(3) and the 1alpha, 25(OH)(2)-24-oxo-D(3) elicit their biological effects by acting through the VDR/VDRE.