The 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) plays an important role in regulating concentrations of both the precursor 25-hydroxyvitamin D3 [25(OH)D3] and the hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3]. Previous studies suggest that Cyp24a1-null mice cannot clear exogenous 1alpha,25(OH)2D3 efficiently. Here, we examined the metabolic clearance in Cyp24a1-null mice in vivo and in vitro using a physiological dose of [1beta-3H]1alpha,25(OH)2D3 or [26,27-methyl-3H]25(OH)D3. Cyp24a1-null mice showed difficulty in eliminating [1beta-3H]1alpha,25(OH)2D3 from the bloodstream and tissues over a 96-h time course, whereas heterozygotic mice eliminated the hormone within 6-12 h, although there was clearance of labeled hormone into water-soluble products involving liver in both genotypes. RT-PCR showed that Cyp24a1-null mice have decreased expression of 25-hydroxyvitamin D-1alpha-hydroxylase that must play a role in their survival. After the administration of [26,27-methyl-3H]25(OH)D3, Cyp24a1-null mice showed higher [26,27-methyl-3H]25(OH)D3 levels and no [26,27-methyl-3H]24,25(OH)2D3 formation, whereas heterozygotic mice showed significant [26,27-methyl-3H]24,25(OH)2D3 production. Based upon in vitro experiments, keratinocytes from Cyp24a1-null mice fail to synthesize [1beta-3H]calcitroic acid from [1beta-3H]1alpha,25(OH2D3 or [26,27-methyl-3H]24,25(OH)2D3 from [26,27-methyl-3H]25(OH)D3 as do control mice, confirming the target cell catabolic role of CYP24A1 in these processes. Finally, the role of vitamin D receptor (VDR) in the vitamin D catabolic cascade was examined using VDR-null mice. Keratinocytes from VDR-null mice failed to metabolize [1beta-3H]1alpha,25(OH)2D3 confirming the importance of vitamin D-inducible, VDR-mediated, C24 oxidation pathway in target cells. These results suggest that the absence of CYP24A1 or VDR retards catabolism of 1alpha,25(OH)2D3 and 25(OH)D3, reinforcing the physiological importance of CYP24A1 in vitamin D homeostasis.