Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (Pi) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH4Cl (acidosis). At killing, blood pH and plasma bicarbonate were 7.36 +/- 0.01 and 21.8 +/- 0.8 mequiv./l, respectively, in control and 7.12 +/- 0.03 (P less than 0.01) and 11.1 +/- 1.2 (P less than 0.01) in acidotic rats. Serum Pi was similar in both groups, while 24 h urine Pi excretion was higher in the acidotic group (P less than 0.01). Peak sodium-dependent uptake of Pi, measured after 1.5 min of incubation, was higher in controls than acidotic rats (4442 +/- 464 vs. 2412 +/- 259 pmol/mg protein, P less than 0.01), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for Pi and glucose uptake were similar in the two groups. Km for Pi uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, Vmax was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits Pi uptake by the brush border of the proximal tubule by decreasing the availability of Pi carriers of the renal brush-border membrane.