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The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood–brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co-application of transporter inhibitors and competing amino acids. Endothelial abluminal L-glutamate uptake was almost abolished by co-application of an EAAT-1 specific inhibitor, whereas luminal uptake was inhibited by L-glutamate and L-aspartate (1 mM). L-glutamate uptake followed Michaelis–Menten-like kinetics with high and low affinity at the abluminal and luminal membrane, respectively. This indicated that L-glutamate is taken up via EAAT-1 at the abluminal membrane and exits at the luminal membrane via a low affinity glutamate/aspartate transporter. Metabolism of L-glutamate and transport of metabolites was examined using [U-13C] L-glutamate. Intact L-glutamate and metabolites derived from oxidative metabolism were transported through the endothelial cells. High amounts of L-glutamate-derived lactate in the luminal medium indicated cataplerosis via malic enzyme. Thus, L-glutamate can be transported intact from brain to blood via the concerted action of abluminal and luminal transport proteins, but the total brain clearance is highly dependent on metabolism in astrocytes and endothelial cells followed by transport of metabolites.