The neutral amino acid carrier composition of luminal and abluminal membranes of the blood-brain barrier has been studied using isolated membrane vesicles. Phenylalanine was carried almost exclusively by a high affinity (Km = 10 +/- 2 microM), Na(+)-independent amino acid transport system, presumably L1 system, that was found to be symmetrically distributed between luminal and abluminal membranes. Inhibition of phenylalanine uptake was used to determine the affinities (Ki values) toward leucine (17 +/- 3 microM), tryptophan (8 +/- 1), 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH) (11 +/- 2), alanine (628 +/- 117), and glutamine (228 +/- 51). Alanine was found to be transported by two Na(+)-dependent transport systems that were located exclusively on the abluminal membrane. Kinetic and inhibition experiments indicated that one of these activities was due to system A, which is probably the main route for Na(+)-dependent alanine transport (Km = 0.6 +/- 0.2 mM) under physiological conditions. The other Na(+)-dependent activity was attributed to a B(o,+)-like system based on its sensitivity toward BCH. This latter system showed greater affinity for large neutral amino acids. The affinities of these two transport systems for several other amino acids were also studied.