Acute administration of dihydroxycholecalciferol [1,25(OH)₂D₃] blunts phosphaturia and increases urinary cAMP excretion in parathyroid hormone (PTH)-infused parathyroidectomized (PTX) rats. Because chronic administration of 1,25(OH)₂D₃ enhances the phosphaturic response to exogenous parathyroid hormone despite blunting of urinary cAMP excretion, we have examined the expression of the renal cortex type II Na-P_i cotransporter (NaPi-2) mRNA and protein in 1) chronic PTX Sabra rats, 2) PTX rats receiving a physiological dose of 1,25(OH)-2-D₃, 3) PTX rats receiving 35 ng/h of PTH, and 4) rats receiving both PTH and 1,25(OH)₂D₃, for 7 days via osmotic minipumps. Our results confirm that there is increased phosphaturia in the PTH+1,25(OH)₂D₃-infused animals despite blunting of urinary cAMP excretion, a reduced filtered load of phosphate, and lack of a phosphaturic effect by 1,25(OH)₂D₃ alone. Both PTH and 1,25(OH)₂D₃ significantly reduced expression of renal cortex NaPi-2 mRNA and NaPi-2 protein, and the administration of PTH together with 1,25(OH)₂D₃ had additive effects in further decreasing NaPi-2 mRNA and NaPi-2 protein levels. Expression of two other epithelial transporters, type 1 Na-sulfate and type 1 Na-glucose cotransporters, were not different between the groups, suggesting specificity of the effects of PTH and 1,25(OH)₂D₃ on phosphate transport. The effect of chronic administration of 1,25(OH)₂D₃ has not been noted previously, and the cellular mechanisms and signaling processes that mediate the decrease in NaPi-2 remain to be determined.