Sodium-dependent transport of inorganic phosphate into brush border membrane vesicles is strongly influenced by altering pH of the incubation medium (pHo). At constant total phosphate concentration an increase in pHo leads to an increase in the uptake of inorganic phosphate. Uptake of inorganic phosphate, however, is not affected by the intravesicular pH (pHi) or by transmembrane pH differences (pHo--pHi). If initial phosphate uptake is studied as a function of total phosphate concentration in the medium the half saturation concentration increases when pHo is raised from 6.3--6.9 but remains unaltered between pHo 6.9 and 7.8 Vmax increases about 3-fold between pHo 6.3 and 6.9 and by a factor of about 1.6 between pHo 6.9 and 7.4. The pHo- dependence of phosphate uptake is diminished by increasing sodium concentrations. Altering transmembrane electrical potential difference by potassium + valinomycin-induced diffusion potentials or by anion replacement fails to demonstrate electrogenicity of sodium-phosphate cotransport. Experiments using a potential-sensitive fluorescent dye, however, indicate a vesicle inside positive electrical potential difference when inorganic phosphate is added. The phosphate-induced alterations in the electrical potential difference are sodium-dependent and more pronounced at low pHo values. Together with earlier observations these results suggest that translocation of inorganic phosphate across the proximal tubular brush border membrane is mediated by cotransport of 2 sodium ions with one either monovalent or divalent phosphate molecule according to its availability in the tubular fluid. The pH sensitivity of this transport system is rather due to alterations in the transport system itself than to pH- dependent alterations in the ratio of monovalent to divalent phosphate.