Intestinal brush border membrane vesicles were prepared from 21-wk-old Holstein bulls. The method of preparation included magnesium precipitation, differential centrifugation and sucrose density gradient centrifugation. Purification of brush border membranes was indicated by sevenfold enrichment of alkaline phosphatase activity. Uptake of D-[U-14C]glucose was sodium-stimulated and exhibited characteristic "overshoot" phenomenon. Sodium-dependent and sodium-independent D-glucose uptake was into an osmotically active space. Phloridzin (100 mM) completely inhibited sodium-dependent, but did not affect sodium-independent transport. Sodium-dependent D-glucose transport was inhibited more by D-glucose than by D-galactose, which inhibited more than D-xylose did. The sodium-dependent D-glucose transport was not inhibited by D-fructose, D-ribose or D-arabinose. Sodium-independent D-glucose transport was unaffected by the sugars tested. Glucose transport in bovine intestinal brush border is similar to that in monogastric species and is composed of two pathways: a sodium-dependent inhibitable system and a diffusional system.