Although the kidney and intestine are among the major organs involved in both the biotransformation and action of vitamin D ₃ , they exhibit very distinct roles in calcium and D ₃ homeostasis. The aim of the present studies was to investigate the relative in vivo responsiveness of renal and intestinal 1,25(OH) ₂ D ₃ -24-hydroxylase (24-hydroxylase) mRNA levels to calcitriol (1,25(OH) ₂ D ₃ ) following acute or chronic 1,25(OH) ₂ D ₃ exposure using hypocalcemic vitamin D-depleted rats as an experimental model. Intestinal 24-hydroxylase mRNA levels were very responsive to a single i.v. injection of 2·4, 12 or 120nmol 1,25(OH) ₂ D ₃ /kg but in kidney the mRNA levels only increased following exposure to the highest 1,25(OH) ₂ D ₃ concentration, and exhibited a maximum response only 30% of that in the intestine despite similar tissue uptake of the hormone. To evaluate whether the kidney might preferentially respond to endogenously produced 1,25(OH) ₂ D ₃ , animals received increasing doses of 25(OH)D ₃ . Although the intestinal 24-hydroxylase transcript was highly induced, the renal transcript was unresponsive to 25(OH)D ₃ treatment despite circulating 1,25(OH) ₂ D ₃ concentrations of 24 nmol/l. By contrast, intestinal 24-hydroxylase mRNA levels were largely unresponsive to long-term calcitriol administration while the renal transcript, although insensitive to a physiological dose, responded to pharmacological 1,25(OH) ₂ D ₃ doses. However, when challenged acutely with 1,25(OH) ₂ D ₃ following chronic exposure, the kidney 24-hydroxylase mRNA levels remained largely unresponsive in contrast to the intestinal transcript which was markedly induced. These data indicate that significant differences exist in the in vivo tissue responsiveness of the 24-hydroxylase mRNA. Indeed, the gene exhibited high intestinal responsiveness to acutely, but not chronically, administered 1,25(OH) ₂ D ₃ , while in the kidney it only responded to high exogenous 1,25(OH) ₂ D ₃ delivered either acutely or chronically. In addition, these site-specific regulatory mechanisms governing the expression of the 24-hydroxylase gene are independent of the endocrine calcium status and render the kidney relatively resistant to endogenously produced 1,25(OH) ₂ D ₃ .