Purified brush border membranes were obtained from homogenized jejunal epithelial cells of cattle by divalent cation aggregation of nonbrush border membranes and differential centrifugation. Membrane marker enzyme assays determined effectiveness of the fractionation procedure. Compared with cellular homogenate, maltase and sodium+-potassium+-adenosine triphosphatase specific activities in the membrane fraction isolated from the interface of discontinuous (35 and 45% wt/wt) sucrose gradients increased 14.5- and 1.9-fold whereas enzyme recoveries averaged 20.2 and 2.4%. These data indicate significant enrichment in brush border membranes with minimal basolateral membrane contamination. Vesicles formed from this membrane fraction had a predominately (93%) luminal side out orientation. After incubating vesicles with radiolabeled substrates, vesicles and accumulated substrates were separated from the incubation buffer by filtration and substrate uptake quantified by liquid scintillation counting. Observed uptake was the result of substrate accumulation within an osmotically active intravesicular space and was not due to nonspecific binding of the substrate to vesicular membranes. Vesicles exhibited sodium-dependent and independent substrate uptake pathways and were able to discriminate between substrate stereoisomers for uptake. Major differences were not detected between results obtained with vesicles prepared from fresh or frozen intestines. These vesicles can be utilized to investigate nutrient uptake by the bovine small intestine.