Uptake of D- and L-glucose, and fructose by purified brush border membrane vesicles isolated from human small intestine was studied using a rapid filtration technique. The uptake of D-glucose by the vesicles was osmotically sensitive and represented transport into an intravesicular space and not binding to the membranes. Transport of both, D- and L-glucose was inhibited by phlorizin. Uptake of D-fructose into the brush border vesicles was not stimulated by sodium. In the presence of a sodium gradient D-glucose was taken up 5 times faster than L-glucose. The amount of D-glucose transported into the vesicles in the presence of a sodium gradient was transiently higher than the amount of D-glucose taken up at equilibrium (over-shoot). D-Glucose transport was stimulated only by a sodium gradient; other monovalent cations had no effect. In the presence of a sodium gradient D-glucose transport was increased by the nearly impermeable anion sulfate as compared with uptake of D-glucose in the presence of a sodium chloride gradient. This indicates an influence of the electrical membrane potential on the sodium coupled non-electrolyte transport.