The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Previous work in this laboratory has indicated that 24,25(OH)2D3 inhibits phosphate uptake in isolated intestinal cells and perfused duodenal loops. It is critical to show this effect in the whole animal to determine the presence of any confounding factors. Studies were therefore undertaken to determine if 24,25(OH)2D3 had a similar effect in vivo. 24,25(OH)2D3 has two isomers which are 24R, 25dihydroxyvitamin D3 [24R,25(OH)2D3] and 24S, 25dihydroxyvitamin D3 [24S,25(OH)2D3]. We studied these two isomers separately and tested them over a time course of 1, 5, 10, 15, and 18 h after steroid using chicks on regular diet, but fasted, and chicks on a lower vitamin D diet. All chicks were anesthetized prior to surgical exposure of the duodenal loop and injection of a solution containing H3 32PO4 into the lumen. An initial time course study of phosphate transport determined that 3 to 9 min of absorption in vivo was in a linear range, as judged by serum levels of radioactivity. Chicks were then injected with either 200 μg of 24R,25(OH)2D3, 20 μg of 24S,25(OH)2D3, or vehicle for control groups within the same time course studies. We found that the isomers had different effects on phosphate absorption. 24R,25(OH)2D3 had a hypophosphatemic effect in vivo. The serum levels of radionuclide revealed hypophosphatemic effects at 1, 5, 15, and 18 h time points with a decrease of 20%, 42%, 39%, and 43%, respectively, (P< 0.05) compared with controls; chicks raised on a low vitamin D diet also showed a decrease in phosphate absorption at 10 h time point by 33%. In contrast, 24S,25(OH)2D3 stimulated intestinal phosphate absorption at the 5-h time point by 64%, but had no other effects at the other time points tested. Because 24S,25(OH)2D3 was largely ineffective, dose-response studies were undertaken with only the 24R,25(OH)2D3 isomer. In comparing phosphate absorption in chicks fasted 18 h, and dosed with vehicle, 100 μg, 200 μg, or 300 μg of steroid 1 h prior to experimentation it was found that the lowest dose increased absorption to 99% of controls, while the 200 μg and 300 μg doses decreased phosphate absorption. (48 pages)