Glutamate transporters belonging to the "excitatory amino acid transporter" (EAAT) family are crucial for controlling the extracellular glutamate concentration and for terminating glutamatergic synaptic transmission. EAAT glutamate transporters are not only secondary-active glutamate transporters, but also conduct a variety of anions in a channel-mediated fashion. At present, the molecular basis of EAAT-mediated anion conduction is insufficiently understood. It is unclear whether permeant anions move through the same transport pathway as glutamate or use a distinct permeation path. We here use fluorescence spectroscopy on solubilized and purified prokaryotic transporter homologue, Glt_Ph from P. horikoshii_, and voltage-clamp fluorometry (VCF) on the neuronal glutamate transporter hEAAT3 expressed in Xenopus oocytes to detect conformational rearrangements that report on binding of anions. An inserted tryptophan (L130W) in Glt_Ph changes its environment upon binding of Na⁺ and aspartate and thus allows quantification of binding by changes of tryptophan fluorescence (see abstract by Ewers, Shcherbyna, Hidalgo and Fahlke, this meeting). Addition of Cl^- results in increased fluorescence at all aspartate concentrations, without changes in aspartate affinity. The effect of Cl^- was abolished by a point mutation reported to reduce anion conductance in Glt_Ph (S65V). We conclude that Cl^- and aspartate do not bind to the same site within Glt_Ph, suggesting separate permeation pathways. To localize the anion permeation pathway and to identify conformational changes associated with anion permeation and gating of the anion channel, we employed voltage-clamp fluorometry on hEAAT3 in Xenopus oocytes. Individual residues were mutated to cysteines, the inserted cysteines were labeled with Alexa Fluor 546 and changes of fluorescence upon perfusion with anions or substrates were monitored under voltage clamp. We identified two cysteines within the transmembrane domain 4 (TM4) (M205C) and the large extracellular loop between TM3 and TM4 (V120C) that report on glutamate binding as well as on anion binding. The relative change in intensity upon substrate application differed for distinct residues. For some of the residues, we could detect voltage-dependent fluorescence intensity changes. The voltage dependency of the fluorescence changes is altered in intensity and shape by the application of glutamate and furthermore differs in response to various external anions (Cl^-> NO₃^-> SCN^-). Our data support the notion that the anion conduction pathway is formed by the amino-terminal half of the transporter protein. Our approach will help identifying residues involved in anion permeation and in opening and closing of the EAAT-associated anion channels.