Summary 1,25-Dihydroxycholecaliferol (1,25-DHCC) is a metabolite of vitamin D produced in the kidney from the major circulating biologically active form of the vitamin, 25-hydroxycholecalciferol (25-HCC) and has been demonstrated to be the derivative of cholecalciferol (CC) primarily responsible for the initiation of calcium transport from the gut contents. Because previous studies have shown that both CC and 25-HCC act acutely to enhance phosphate, sodium and calcium reabsorption in the kidney, the effect of 1,25-DHCC was evaluated in clearance studies performed in mildly expanded thyroparathyroidectomized dogs. In a dose of 25 U (0.625 μg), 1,25-DHCC induced an invariable fall in both fractional and absolute phosphate excretion (mean declines of 40 and 36%, respectively, p < .05 and < .01). Fractional sodium and calcium excretion also deccreased by mean values of 26 and 36%, respectively (p < .02). Renal hemodynamic changes and alterations in serum ultrafilterable calcium and phosphate concentrations did not occur, providing evidence for a direct tubular effect of the metabolite. A comparison of the action of 1,25-DHCC on phosphate excretion, to that obtained previously with CC and 25-HCC revealed the following: First, whereas the former metabolite further shortened (to 20–30 min) the time required for the onset of the renal action of CC (50–60 min) and 25-HCC (30–40 min), it did not act immediately; and secondly, an equivalent dose of 25-HCC had a greater effect on phosphate excretion than did 1,25-DHCC in this investigation.