1,25-dihydroxyvitamin D3 [1,25(0H)2D3] has been shown to promote phosphate transport rapidly in the perfused duodenal loop, relative to controls, reaching treated/av basal at T = 40 min = 1.82 ± 0.42 and 1.11 ± 0.21, respectively. By using isolated chick enterocytes, studies were undertaken to determine whether 1,25(0H)2D3 has a direct effect on isolated intestinal cells that is manifested by either enhanced uptake or extrusion of phosphate. In time course studies, with 4- to 8-wk-old chicks, 32P uptake in enterocytes at 10 min after addition of test substance was 0%, 130%, 151%, and 123% of controls for 10 pM, 50 pM, 130 pM, and 300 pM 1,25(0H)2D3, respectively. The metabolite 24,25- dihydroxyvitamin D3 [24,25(0H)2D3] exerted an inhibitory effect on phosphate uptake by 1,25(0H)2D3 at a concentration of 130 pM. This result was in agreement with perfusion studies and supports the physiological relevance of isolated cell studies. For signal transduction studies, isolated enterocytes were incubated with 20 µM forskolin (an activator of protein kinase A), 100 nM phorbol ester (an activator of protein kinase C), or 2 µM BAY K 8644 (a calcium channel activator). Enhanced 32P levels relative to controls were found for phorbol ester (126% of controls at T = 7 min, P < 0.05) and BAY K 8644 (150% of controls at T = 7 min, P < 0.05) but not for forskolin, suggesting involvement of protein kinase C and calcium channel signal transduction pathways in uptake. These results paralleled those observed for the perfused duodenal loop. For aging studies, white leghorn roosters were raised for 7, 14, and 28 wk prior to experiments. These studies showed a 1,25(0H)2D3-mediated increase in 32P uptake in isolated cells at 7 wk, but not at 14 or 28 wk. Further analysis of isolated basal lateral membrane (BLM) on SDS-PAGE followed by Western analysis with a well characterized antibody (Ab099) showed a decreased expression of the putative membrane receptor for 1,25(0H)2D3 with increasing age, paralleling the results obtained for 32P uptake in isolated intestinal cell studies. Analyses of 1,25(0H)2D3 effect on protein kinase C activity likewise revealed hormone-mediated stimulation in cells from 7-wk- old chicks, with decreasing responsiveness at a later age. The combined results indicate a physiologically important role for 1,25(0H)2D3 membrane-initiated phosphate uptake in enterocytes of young, rapidly growing animals. Furthermore, these studies validate the use of isolated intestinal cells for further studies on ribozyme-mediated ablation of the 1,25(0H)2D3 membrane receptor function.