Brush border membrane vesicles (BBMV) were prepared from eel (Anguilla anguilla) intestine by a Mg-ethylene-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid precipitation technique; the BBMV were enriched 16, 12, and 13 times in leucine aminopeptidase, maltase, and alkaline phosphatase activities with respect to the starting mucosal scraping. D-[3H]glucose and L-[3H]alanine transport by these vesicles was studied by a rapid filtration technique. D-Glucose uptake was stimulated by a transmembrane Na gradient but not by an identical Na gradient in the presence of phloridzin or by a choline gradient. The Na-dependent D-glucose uptake was increased by rendering the vesicle interior electrically negative, suggesting electrogenic cotransport of the sugar with Na+. Kinetic analysis gave an apparent affinity constant (Kapp) of 0.20 mM and maximal rate (Jmax) of 6.87 nmol X mg protein-1 X min-1 for glucose influx in the presence of a Na gradient. In addition, a significant apparent diffusional permeability of these membranes to glucose (1.41 microliters X mg protein-1 X min-1) was observed. L-Alanine uptake in eel BBMV was shown to occur via 1) saturable Na-dependent pathway (Kapp = 1.29 mM, Jmax = 3.61 nmol X mg protein-1 X min-1), 2) a saturable Na-independent pathway (Kapp = 0.59, Jmax = 1.49), and 3) a nonsaturable component representing apparent diffusion (permeability coefficient P = 0.57 microliter X mg protein-1 X min-1). These findings suggest that similar transport systems for glucose and alanine are found in the fish and mammalian intestinal brush border membrane.