Calcitriol (CT) stimulates Na+-P(i) cotransport in a subclone of opossum kidney cells (OK-7A) by a genomic mechanism. An experimental model of renal proximal tubular cells in which CT affects Na+-P(i) cotransport would be useful for examining the mechanisms of this effect. This study evaluated the effect of CT on Na+-P(i) cotransport in opossum kidney (OK) cells. CT had no effect on Na+-P(i) cotransport in wild-type OK cells and in the OK-B, OK-H, and OK-P subclones. In contrast, CT at physiological concentrations stimulated Na+-P(i) cotransport in the OK-7A subclone; the effect was dose related with a 52% increase at 10-11 M CT, as well as a maximal twofold stimulation at 10-9 M. CT (10-11 M) increased the maximum uptake for Na+-P(i) cotransport (V(max) = 3.55 ± 0.16 vs. 2.51 ± 0.17 nmol · mg protein-1 · 5 min-1, P < 0.01), without affecting the apparent Michaelis constant (K(m) = 30.6 ± 1.0 vs. 30.8 ± 0.7 μM). The stimulatory effect on Na+-P(i) cotransport was specific for CT and did not occur with 25- hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or 1β,25-dihydroxyvitamin D3. At 10-11 M CT, the stimulation of Na+-P(i) uptake in OK-7A cells was maximal at 3 h; it was completely abolished by preincubation with actinomycin D or cycloheximide. Both calphostin C, an inhibitor of protein kinase C (PKC), or prolonged incubation with phorbol 12-myristate 13-acetate, to downregulate the PKC pathway, partially inhibited the stimulatory effect of CT on Na+-P(i) cotransport in OK-7A cells. The stimulatory effect of CT on Na+-P(i) cotransport persisted in the presence of parathyroid hormone (PTH) and was additive to the stimulatory effect of phosphate depletion. Receptor binding studies suggested the presence of vitamin D receptors in all the OK subclones, raising the possibility of postreceptor differences preventing the stimulatory effect on Na+-P(i) cotransport. These studies suggest that CT in physiological concentrations stimulates Na+-P(i) cotransport in OK-7A cells by a mechanism that requires DNA transcription and protein synthesis and is modulated by the PKC pathway. Moreover, this effect is additive to the stimulatory effect of phosphate depletion and persists in the presence of PTH inhibition. Thus the OK-7A cells are a useful model for investigating the mechanism by which CT stimulates Na+-P(i) cotransport in an established renal epithelial cell line.