Phosphate transport was studied in isolated brush-border membrane vesicles prepared from human jejunum. Transport consisted of two components: passive diffusion across the brush border, and sodium-dependent, carrier-mediated uptake. Both carrier-mediated transport and passive diffusion of phosphate were significantly greater at pH 6.1 than at pH 7.4. Sodium-dependent uptake was saturable at both pH values; the apparent Km was not affected by pH, whereas Vmax was significantly greater at pH 6.1 (179.3 vs. 118.4 pM/mg per 15 s). Phosphate uptake was half-maximally stimulated by a sodium concentration of 34 mM at pH 7.4, and a sodium concentration of 76 mM at pH 6.1. At both pH values, Hill coefficients were greater than 1 and were unaffected by pH. Moreover, sodium-dependent phosphate uptake was electroneutral at pH 7.4 and electrogenic at pH 6.1. These data suggest that at acid and neutral pH, both monovalent and divalent phosphate are transported across the human jejunal brush border membrane. The transport of each phosphate molecule is associated with the cotransport of two sodium ions.