The 25(OH)D₃/1,25(OH)₂D₃ 24‐hydroxylase (24‐hydroxylase) displays an induction profile responsive to vitamin D (D) abundance and is hence only observed in normal extracellular Ca²⁺ concentrations. However, the participation of Ca²⁺ in the expression of the 24‐hydroxylase gene in vivo is not known. The present studies investigate the role played by the circulating Ca²⁺ and the D₃ and/or 1,25(OH)₂D₃ status on the 1,25(OH)₂D₃‐mediated inducibility of the 24‐hydroxylase gene in rat duodenum. Hypocalcemic D‐depleted rats were supplemented with calcium alone to normalize serum Ca²⁺ without normalizing the D₃ status or were acutely or chronically supplemented with D₃ or 1,25(OH)₂D₃. Messenger RNA for the 24‐hydroxylase was undetectable in the intestine of hypocalcemic D‐depleted rats, and short‐ or long‐term calcium supplementation was completely unsuccessful in inducing its expression. By contrast, acute 1,25(OH)₂D₃ administration led to significant increases in the levels of expression of the gene which was independent of the calcium intake, the prevailing circulating Ca²⁺, and the D₃ or 1,25(OH)₂D₃ status. Moreover, 24‐hydroxylase gene expression was only found to respond to acutely administered 1,25(OH)₂D₃, the mRNA levels being unaltered following continuous exposure to physiological or pharmacological doses of the hormone for 7 days. Time‐course studies revealed, however, that induction of the gene was clearly apparent early in the 1,25(OH)₂D₃ supplementation course but gradually faded by 3 days to return to basal uninduced levels by 7 days, suggesting the presence of intestinal adaptation mechanism(s) which down‐regulated the responsiveness in the continuous presence of 1,25(OH)₂D₃. Our data show the lack of effect of calcium alone or in combination with 1,25(OH)₂D₃ on the in vivo induction of the 24‐hydroxylase gene expression in rat intestine. By rapidly reacting to surges in 1,25(OH)₂D₃ concentrations, the 24‐hydroxylase efficiently controls the amount of 1,25(OH)₂D₃ in intestine as the first step in the biotransformation pathway aimed at the irreversible clearance of the secosteroid hormone. (J Bone Miner Res 1995;10:1148–1157)