Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodiumdependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na + -dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na + -dependent glutamate/aspartate transporter and Na + -dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover. Keywords: Bergmann glia, glutamate transporters, glutamate/ glutamine shuttle, glutamine transporters. EAAT 1-5, excitatory amino acid transporters; ECL, enhanced chemiluminescence reagent; EDTA, ethylenediaminetetraacetic acid; GLAST, Na + -dependent glutamate/aspartate transporter; Gln, glutamine; Glt-1, glutamate transporter-1; GS, glutamine synthetase; KA, kainate; KBP, kainate binding protein; L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; L-Glu, L-glutamate; NMDA, N-methyl-D-aspartate; NP-40, Nonidet P-40; PBS, phosphate-buffer saline; PBT, phosphate-buffer saline containing 0.3% Triton; SDS-PAGE, sodium dodedecyl sulfate polyacrylamide gel electrophoresis; SNAT2-3-5, Na + -dependent neutral amino acid transporter 2-3-5; TBS, Tris-buffered saline; THA, threo-b-hydroxyaspartate; trans-ACPD, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxilic acid.