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)2D ₃. 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,25-dihydroxyvitamin D₃ [24R,25(OH)₂D ₃] and 24S,25-dihydroxyvitamin D₃ [24S,25(OH)₂D ₃]. We studied these two isomers separately and tested them over a time course of 1, 5, 10, 15 and 18 hours 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 H₃³²PO₄ 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 24R,25(OH) ₂D₃, 20 µg 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)₂D ₃ had a hypophosphatemic effect in vivo. The serum levels of radionuclide revealed hypophosphatemic effects at 1, 5, 15 and 18 hours time points with a decrease of 56%, 42%, 39% and 43%, respectively, (P< 0.05) compared with controls. In contrast, 24S,25(OH)₂D₃ stimulated intestinal phosphate absorption at the five hour time point by 64%, but had no other effects at the other time points tested.