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25-Hydroxyvitamin D3[25(OH)D3] is a metabolite of vitamin D3 that has long been considered to be an inactive precursor of the hormonally active metabolite 1,25- dihydroxyvitamin D3[l,25(OH)2D3]; consequently very few studies have addressed the potential biological activity of 25(OH)2D3. However, it is known that 100 nM 25(OH)2D3 increases calcium transport in the perfused duodenal loop of the chicken to 200% of controls within 20 minutes. The hypothesis of the current study is that 25(OH)2D3 may be a hormonally active metabolite and its effects can be studied in isolated chick enterocytes. To begin testing this postulate, time course studies of 45Ca uptake were undertaken in isolated intestinal cells (from 7 wk chicks). After establishing the basal uptake of 45Ca for 5 minutes, cells were treated with vehicle(< 0.01% v/v ethanol, final concentration) or 25 nM, 50 nM, 100 nM, or 300 nM 25(OH)2D3 and samples were taken at T = 1, 3, 5, 7, and 10 min. With increasing concentrations of steroid, the enterocyte 45Ca decreased. The optimal concentration of 100 nM 25(OH)2D3 induced the most rapid response: within 1 min 45Ca decreased to 54% of controls (P < 0.001) and 70% of the controls at T= 3, 5, and 7 min (P < 0.01 to < 0.05, relative to controls). Comparison of the 7- min time points for 25 nM, 50 nM, 100 nM, and 300 nM 25(OH)2D3 appeared to yield a biphasic dose response curve with values of 45Ca observed at 99% (NS, not significant), 75% (P < 0.05), 70%(P < 0.01%), and 80% (NS) of corresponding controls, respectively. Physiological levels of 24,25(OH)2D3 (6.5 nM) inhibited the action of 100 nM 25(OH)2D3 in isolated chick enterocytes. Time course studies with isolated enterocytes from 14 wk and 28 wk chickens treated with 100 nM 25(OH)2D3 also showed decreased responsiveness: at T= 1 min 45Ca levels in 7 wk, 14 wk, and 28 wk were 54% (P < 0.01), 83% (NS), and 80% (NS) of corresponding controls, respectively. Experiments with the calcium channel activator BAY K8644 (2 μM) and protein kinase A (PKA) activator forskolin (20 μM) revealed enhanced levels of 45Ca at T= 10 min that were 132% and 140% of corresponding controls, respectively (each, P < 0.05). Phorbol ester treatment of the cells resulted in significant increases in the levels of 45Ca between the treated cells and corresponding controls at T=7 and 10 min. Cells treated with 100 nM 25(OH)2D3 revealed 89.8% and 78.4% increases above controls in PKA activity at T =1 min (P < 0.05) and T=3 min, relative to corresponding controls. However, there was no evidence for the activation of PKC by 25(OH)2D3 during the time period studied.