Hormonal regulation of calcium (Ca) absorption was investigated in a cholecalciferol (vitamin D(3))-supplemented group (hVitD) vs. a control group (cVitD) of growing Great Danes (100 vs. 12.5 micro g vitamin D(3)/kg diet). Although Ca intakes did not differ, fractional Ca absorption was significantly lower in the hVitD group than in the cVitD group. There were no differences in plasma concentrations of Ca, inorganic phosphate, parathyroid hormone, growth hormone or insulin-like growth factor I between groups. Plasma 25-hydroxycholecalciferol [25(OH)D(3)] concentrations were maintained in the hVitD dogs at the same levels as in the cVitD dogs due to increased turnover of 25(OH)D(3) into 24,25-dihydroxycholecalciferol [24,25(OH)(2)D(3)] and 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)]. In hVitD dogs, the greater plasma 24,25(OH)(2)D(3) concentration and the enhanced metabolic clearance rate (MCR) of 1,25(OH)(2)D(3) indicated upregulated 24-hydroxylase activity. The increased MCR of 1,25(OH)(2)D(3) decreased plasma 1,25(OH)(2)D(3) concentrations. In hVitD dogs, the greater production rate of 1,25(OH)(2)D(3) was consistent with the 12.9-fold greater renal 1alpha-hydroxylase gene expression compared with cVitD dogs and compensated to a certain extent for the accelerated MCR of 1,25(OH)(2)D(3). The moderately decreased plasma 1,25(OH)(2)D(3) concentration can only partially explain the decreased Ca absorption in the hVitD dogs. Intestinal vitamin D receptor concentrations did not differ between groups and did not account for the decreased Ca absorption. We suggest that 24,25(OH)(2)D(3) may downregulate Ca absorption.