L-Glutamate (L-Glu) is known to be a relaxant of pericytes and to induce changes in microcirculatory hemodynamics. Since the concentration of L-Glu which induces the dilation of retinal capillaries is reported to be high compared with the estimated concentration in the retinal interstitial fluid, it is hypothesized that some systems involving concentrative L-Glu release are present in retinal pericytes. The purpose of this study was to investigate the existence of L-Glu-storing systems, which contribute to autocrine L-Glu release, in retinal pericytes using conditionally immortalized rat retinal pericytes (TR-rPCT1 cells), which express mRNAs of L-Glu-synthesizing enzymes from L-glutamine (L-Gln). TR-rPCT1 cells express the mRNAs of vesicular L-Glu transporter 1 (VGLUT1), indicating that L-Glu in the cytoplasm is taken up into VGLUT1-expressing vesicles of retinal pericytes. L-Glu and L-Gln are taken up into TR-rPCT1 cells via Na⁺-dependent saturable process(es) with a K _m value of 22.4 µM and 163 µM, respectively. The [³H]L-Glu uptake was inhibited by ca. 50% in the presence of D-aspartate, a substrate of excitatory amino acid transporter (EAAT) subtypes, whereas substrates of alanine–serine–cysteine transporter (ASCT) subtypes exhibited only a weak inhibitory effect on [³H]L-Glu uptake compared with D-aspartate. Regarding the L-Gln uptake by TR-rPCT1 cells, the inhibitory effect of ASCT substrates on the [³H]L-Gln uptake was stronger than that of substrates of other neutral amino acid transport systems. Consequently, it was determined that EAAT1 and ASCT2 play a role in the transport of L-Glu and L-Gln, respectively, from retinal interstitial fluid to the cytoplasm of retinal pericytes.