The purpose of this study was to elucidate the transport mechanism(s) of L-glutamate (L-Glu), a neuroexcitatory neurotransmitter, in the inner blood-retinal barrier (BRB). The L-Glu transport was evaluated by an in vitro uptake study with a conditionally-immortalized rat retinal capillary endothelial cell line, TRiBRB2 cells. L-Glu uptake by TR-iBRB2 exhibited time-and concentration-dependence, and was composed of high-and low-affinity processes with Michaelis-Menten constants (K m ) of 19.3 µM and 275 µM, respectively. Under Na -free conditions, L-Glu uptake by TR-iBRB2 involved one-saturable kinetics with a K m of 190 µM, which is similar to that of the low-affinity process of L-Glu uptake under normal conditions. Moreover, substrates/inhibitors of system X c , which is involved in blood-to-retina transport of compounds across the inner BRB, strongly inhibited the L-Glu uptake under Na -free conditions, suggesting that Na -independent lowaffinity L-Glu transport at the inner BRB is carried out by system X c . Regarding the Na -dependent high affinity process of L-Glu transport at the inner BRB, L-Glu uptake by TR-iBRB2 under normal conditions was significantly inhibited by substrates/inhibitors of excitatory amino acid transporter (EAAT) 1-5, but not alanine-serine-cysteine transporters. Reverse-transcription polymerase chain reaction (RT-PCR) analysis and immunoblot analysis demonstrated that mRNA and protein of EAAT1 are expressed in TR-iBRB2 cells, whereas mRNAs and/or proteins of EAAT2-5 are not. Immunohistochemical analysis revealed that EAAT1 protein is localized on the abluminal membrane of the retinal capillaries. In conclusion, EAAT1 most likely mediates Na -dependent high-affinity L-Glu transport at the inner BRB and appears to take part in L-Glu elimination from the retina across the inner BRB.Key words excitatory amino acid transporter 1; inner blood-retinal barrier; L-glutamate L-Glutamate (L-Glu) is known as an excitatory neurotransmitter. In the retina, it has been reported that L-Glu controls the synaptic transmission between the photoreceptor and the bipolar/horizontal cells. 1) Excess L-Glu over-stimulates its receptors, such as N-methyl-D-aspartate receptors, and then causes neuronal cell death.2) In some case of glaucoma, it has been proposed that L-Glu-mediated neuro-excitotoxicity is involved in the progression of the disease.3) Hence, it is conceivable that some regulatory mechanisms of L-Glu concentration in the retina are present.The blood-retinal barrier (BRB) segregates the circulating blood from retinal interstitial fluid and regulates the exchange of compounds between the circulating blood and the retina. Regarding L-Glu transport across the BRB, Törnquist et al. have shown that in vivo blood-to-retina transport of [ 14 C]LGlu was not significantly inhibited, but increased 1.7-fold, by the co-administration of 10 mM unlabeled L-Glu.4) This result implies carrier-mediated efflux transport of L-Glu across the BRB. It has been shown that retinal capillary endothelial cells form the in...