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 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. Previous studies suggest that Cyp24a1-null mice cannot clear exogenous 1α,25(OH)₂D₃ efficiently. Here, we examined the metabolic clearance in Cyp24a1-null mice in vivo and in vitro using a physiological dose of [1β-³H]1α,25(OH)₂D₃ or [26,27-methyl-³H]25(OH)D₃. Cyp24a1-null mice showed difficulty in eliminating [1β-³H]1α,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-1α-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 [1β-³H]calcitroic acid from [1β-³H]1α,25(OH)₂D₃ 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 [1β-³H]1α,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 1α,25(OH)₂D₃ and 25(OH)D₃, reinforcing the physiological importance of CYP24A1 in vitamin D homeostasis.