To investigate the mechanism of the renal tubular transport effects of 1,25-dihydroxyvitamin D3 (1,25-D3), brush border membrane vesicle inorganic phosphate (Pi) uptake and gluconeogenesis were measured in renal cortex obtained from vitamin D deficient thyroparathyroidectomized rats which had been infused with 6 pmol/h of 1,25-D3 and 0.2 U/h of parathyroid hormone (PTH), a nonphosphaturic ('permissive') dose, for 6 h. When compared to vitamin D deficient animals receiving the vehicles alone, there was a significant increase in Pi transport by those vesicles obtained from the 1,25-D3-treated animals. Substrate-stimulated gluconeogenesis in thyroparathyroidectomized vitamin D deficient animals was decreased in animals which received the 1,25-D3 plus PTH combination. In all groups receiving 1,25-D3 plus PTH, there was a significant decline in urinary Pi excretion. We, therefore, conclude that 1,25-D3 promotes proximal tubular Pi reabsorption through an effect that occurs at the brush border membrane. Furthermore, the administration of 1,25-D3 causes a reduction in renal cortical gluconeogenesis. The administration of a permissive dose of PTH is necessary for these effects to occur.