To evaluate the role of vitamin D in the physiologic response to phosphorus depletion (P depleton) and the response to vitamin D administration in P depletion, we studied vitamin D-deficient (−D) rats, fed either a normal or low phosphorus diet and then injected intraperitoneally on alternate days with replacement vitamin D₃, 1.25 μg qod (D₃); 1.25-dihydroxy-vitamin D₃[1,25(OH)₂D₃] in physiologic, 54 ng qod (LD), and pharmacologic doses, 400 ng qod (HD); or vehicle alone (−D). The following results were obtained: (a) With P depletion, urinary excretion of inorganic phosphorus (Pi) fell to almost undetectable levels in −D rats, and two physiologic features of P depletion a calcemic effect and hypercalciuria, ensued. (b) With administration of vitamin D₃ or 1,25(OH)₂D₃ in either doses to P-depleted rats, the renal retention of Pi was unaltered despite a significant elevation of serum Pi. (c) The calcemic response to P depletion was accentuated by vitamin D sterols, and the hypercalciuria of P depletion was reduced by 1,25(OH)₂D₃, HD > LD > D₃. (d) In −D animals receiving normal Pi (+P), D₃, and 1,25(OH)₂D₃, both LD and HD produced a significant calcemic and phosphatemic effect. (e) Urinary Pi excretion in +P animals was reduced slightly by vitamin D₃ whereas 1,25(OH)₂D₃, both LD and HD, lowered urinary Pi markedly despite an increased serum Pi. (f) The serial values of serum Ca and Pi and urinary Ca in PD rats and the sequential values for urinary and serum Pi in +P rats indicated more rapid effects of 1,25(OH)₂D₃, both HD and LD, compared with D₃. We conclude that: (a) The renal adaptation and physiologic response to PD does not require the presence of vitamin D. (b) 1,25(OH)₂D₃ may directly enhance the renal tubular reabsorption of Pi even as serum Pi rises. (c) A hypocalciuric action of 1,25(OH)₂D₃ in rats on low phosphorus diet could be direct or occur as a consequence of an increase in serum Pi produced by 1,25(OH)₂D₃. The different sequential renal response to D₃ compared with 1,25-(OH)₂D₃ raises the possibility that other natural forms of vitamin D₃ [i.e., 25(OH)D₃, 24,25(OH)₂D₃, etc.] which may be present in vitamin D-fed rats but not those given only 1,25(OH)₂D₃, could modify the actions of 1,25(OH)₂D₃.