Brush-border membrane (BBM) vesicles were prepared from the intestine of yellow eels (Anguilla anguilla) by selective precipitation of all other cellular components. The activity of a Na-substrate cotransporter was evaluated by measuring the rate of decay of an inside negative membrane potential with a voltage-sensitive fluorescent dye; the rate of fluorescence quenching decay in the presence of Na was subtracted from the rate of decay in the presence of both cosubstrates. The kinetics of Na-dependent amino acid transport systems were then determined for the neutral amino acids and their N-methylated analogues. It was concluded that N-methylglycine, N-methylalanine and α-(methylamino)-isobutyric acid are transported by the Na-dependent carrier responsible for proline translocation, which is distinct from the one responsible for glycine, alanine and α-aminoisobutyric acid translocations.