Vitamin D compounds regulate PTH at the transcriptional level, presumably via binding to the vitamin D receptor (VDR), but the exact mechanism is presently unclear. We recently reported that the several vitamin D prohormones with low VDR affinity suppressed PTH, even when their activation was inhibited, raising the possibility that their actions may be VDR independent. To test this hypothesis, we developed a novel organ culture that allowed the assessment of activities of the prohormones on PTH release from wild-type and VDR-null thyroparathyroid explants. The cultures remained viable with respect to PTH release for at least 2 weeks. Full suppression of PTH by the native vitamin D hormone, 1α,25-dihydroxyvitamin D(3) [1α,25 (OH)(2)D(3)], required 2 days, consistent with a transcriptional mechanism, and was reversible, indicating that reduced PTH was not attributable to cell death. Inhibition of PTH release by 1α,25 (OH)(2)D(3) and two prohormones, 25-hydroxyvitamin D(3) and 1α-hydroxyvitamin D(2), was observed in explants from wild-type mice but not in those from VDR-null mice. These findings 1) are the first direct demonstration of the role of the VDR in regulation of PTH by 1α,25(OH)(2)D(3), 2) confirm that the suppressive actions of the vitamin D prohormones are mediated by the VDR, and 3) introduce a novel organ culture model that allows the ex vivo study of the function of parathyroid glands from transgenic animals.