Uptake of inorganic sulfate into brush-border membrane vesicles isolated by a calcium precipitation method from rat small intestine was investigated using a rapid filtration technique and 35Sulfur acid as tracer. Sulfate uptake by membrane vesicles was osmotically sensitive, suggesting transport into an intravesicular space rather than binding to or incorporation into the membrane. Transport of sulfate into brush-border vesicles isolated from rat ileum was only stimulated by sodium ions as compared with other monovalent cations. A typical "overshoot" phenomenon was observed in the presence of an inwardly directed NaCl gradient. Tracer sulfate exchange was faster in the presence of sodium than in the presence of potassium. Addition of the ionophores for monovalent cations, monactin, or gramicidin D, decreased the sodium gradient-driven sulfate uptake. Sulfate uptake showed a saturation phenomenon only in the presence of sodium. Transstimulaton of sodium-dependent sulfate transport was shown with MoO4(2-), but not with PO4(2-) and WO4(2-). Changing the electrical potential difference across the membrane vesicles by establishing different diffusion potentials (anion replacement; potassium gradient +/- valinomycin) did not alter sodium-dependent sulfate uptake. Stimulation of sulfate transport by sodium was greater in membrane vesicles from ileal segments than from duodenum or jejunum. It is concluded that isolated brush-border membranes of rat ileum contain an electroneutrol sodium-sulfate cotransport system.