Brush-border membrane vesicles (BBMV) were prepared from European eel (Anguilla anguilla) intestinal epithelium by a magnesium-ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) precipitation technique. Amino acid transport by these purified vesicle preparations was investigated using either radiolabeled substrates or the voltage-sensitive fluorescent dye 3,3'-diethylthiadicarbocyanine iodide [DiSC2(5)]. All amino acids tested exhibited carrier-mediated, Na+-dependent and Na+-independent transfer processes plus diffusion. The only exceptions were glutamic acid and proline, which displayed Na+ dependency and diffusion but did not appear to be transported by Na+-independent agencies. Carrier-mediated transport kinetic constants (Kapp and Jmax) for several amino acids are reported. Cis-inhibition experiments suggested the presence of at least four distinct Na+-dependent transport systems in eel intestinal BBMV: 1) an anionic transport process for glutamic and aspartic acids; 2) a cationic mechanism for lysine and arginine; 3) a relatively specific neutral amino acid carrier for proline and alpha-(methylamino)isobutyric acid; and 4) a nonspecific neutral amino acid system for most other substrates of this group. This scheme for carnivorous fish intestine most closely approximates that reported for mammalian gut with minor dissimilarities that may relate to metabolic differences or specific dietary requirements of the two vertebrate groups.