The L-system amino acid transporter on the RBE4 cell line, a well established in vitro model of the blood-brain barrier (BBB), was characterised with the aim to evaluate this in vitro BBB model as tool for the systematic exploration of this endogenous carrier system for drug delivery to the CNS. Transport of L-[3H]-leucine in RBE4 cells was rapid, Na+-independent, bidirectional and followed the principles of trans-stimulation. The inhibition profile of L-leucine uptake was consistent with transport mediated by the L-system amino acid carrier with strong inhibition by large neutral amino acids (LNAA) such as L-phenylalanine and 2-aminobicyclo-heptanecarboxylic acid (BCH), whereas small neutral, basic and acidic amino acids had no significant effect. The transport of L-leucine into the RBE4 cells was saturable and followed single carrier Michaelis-Menten kinetics with Km 107 +/- 10 microM. Vmax 9.13 +/- 0.45 nmol/min/mg protein and KD 1.36 +/- 0.13 microl/min/mg protein. The kinetic constants of L-leucine transport, as well as the ranking of the kinetic constants of the transport of other LNAA investigated, correspond to those of the BBB in vivo. The characteristics of the LNAA transport in RBE4 cells suggest that transport is mediated by a system with characteristics similar to the L1 subtype of amino acid transporter, with carrier specificity equivalent to the L1 carrier system at the BBB in vivo. The study shows that the RBE4 cell line is a very suitable tool for the detailed examination of structure-transport relationships with respect to carrier-mediated drug delivery to the CNS via the L-system amino acid carrier at the BBB. The strength of this in vitro BBB model lies in the combination of the advantages of a cell line, being inexpensive, reproducible and easy to maintain, with the brain endothelium-specific expression of transport systems, to produce an efficient assay for the screening of potential neuropharmaceuticals targeted to specific transport routes to enhance CNS drug delivery.