Jejunal epithelial cells from slaughtered Holstein cows were fractionated to obtain purified brush border membranes from which membrane vesicles were prepared for use in amino acid uptake studies. Uptake of alanine was determined by incubation of vesicles with a solution containing radiolabelled alanine, isolation of vesicles and accumulated alanine by filtration, and detection of accumulated alanine by liquid scintillation counting. Uptake studies were conducted under conditions shown to provide linear rates of accumulation. Sodium-dependent active transport was determined as the difference between uptake measured in the presence and absence of sodium in the extravesicular buffer. Inhibition of alanine uptake increased with increasing extra-vesicular inhibitor concentration until a plateau value was reached. Inhibition of sodium-dependent alanine uptake by 100 mM glycine was 72%; 25 mM isoleucine, valine, or methionine completely inhibited initial alanine uptake. These results indicate the existence of at least two sodium-dependent transport systems, one capable and one incapable of accepting glycine for transport. At concentrations designed to represent expected concentrations of free amino acids in intestinal digesta, several equimolar mixtures (.2 to 5 mM) of 20 amino acids inhibited alanine uptake, suggesting that significant interaction among amino acids for uptake may be occurring under in vivo conditions.