The amino-acid transport systems have been characterized in brush-border membrane vesicles prepared from guinea-pig small intestine. Uptake of all amino acids tested was measured at the initial velocity for 5 s. L-Proline, alpha-(methylamino)isobutyrate, glycine, L-alanine and L-methionine were transported dependent solely on an Na+ gradient from the outside to the inside of the vesicles, and L-cysteine, L-phenylalanine and L-leucine were transported dependent largely on the Na+ gradient with a small fraction of Na+-independent transport. The transport of L-aspartic acid and L-lysine was independent of the Na+ gradient and L-lysine transport was somewhat inhibited by the presence of cations, including Na+, K+ and Li+. A cross-inhibition study of the uptake of these amino acids in the brush border of guinea-pig intestine revealed the presence of at least three Na+-dependent and three Na+-independent carrier-mediated systems. One Na+-dependent system interacted mainly with imino acid. Another Na+-dependent system interacted with neutral amino acids, while a third system was selective for glycine. One Na+-independent system is for acidic amino acids, another is responsible for neutral amino acids and a third for cationic amino acids. These transport systems of amino acids in guinea-pig small intestine are compared with those in rabbit and mouse intestine.