To evaluate the mode and tubular sites of action of PO4 administration on renal Ca reabsorption, micropuncture and clearance experiments were performed on acutely parathyroidectomized rats: (1) control (n=7), 2.5% saline expansion + furosemide infusion (0.5 mg/kg/hr); (2) PO4 infusion (n=6), treated as were the controls plus intravenous neutral PO4 (25 mg/kg/hr). CaCI2 was infused to keep plasma UF Ca comparable in both groups [2.68 +/- 0.08 mEq/l (mean +/- S.E.) vs 2.52 +/- 0.10, N.S.]. Po4 infusion increased plasma PO4 (14.0 vs 8.4 mg/dl, p less than 0.001) and FE PO4 (9.03% vs. 0.09%, p less than 0.001) but decreased FE Ca (1.83% vs. 5.95%, p less than 0.02) while GFR and FE Na were similar. FD of Na, PO4 and Ca were similar in late proximal and ED tubule punctures. In the LD tubule, PO4 infusion increased FD PO4 (18.5% vs. 7.9%, p less than 0.005) and reduced FD Ca (3.51% vs. 6.66%, p less than 0.01) and (TF/UF) Ca/Na (0.69 vs. 1.40, p less than 0.001). LD tubule FD Ca (y) was inversely related to TF/P PO4 concentration (X) in LD tubules: (y=7.2 - 0.12x, r=0.68, p less than 0.02). Significant Ca reabsorption (1.68% of filtered load) was observed beyond the superficial LD tubule only during PO4 infusion. These data indicate that phosphate infusion enhances Ca reabsorption independently of PTH, plasma Ca, and renal Na handling at a site within and possible beyond the distal convoluted tubule of the superficial nephron. (J Lab Clin Med 99:317, 1982).