Excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft to ensure low resting glutamate concentrations and to terminate glutamatergic synaptic transmission. EAATs mediate a stoichiometrically coupled transport of glutamate, 3 Na⁺ and 1 H⁺, in countertransport with 1 K⁺. We here study sodium binding to a bacterial EAAT paralog with known structure, Glt_Ph from Pyrococcus horikoshii, using fluorescence spectroscopy. We expressed mutant transporters with an inserted tryptophan in the III-IV interlinker, L130W Glt_Ph and measured substrate-dependent changes in fluorescence of solubilized and purified protein in order to quantify aspartate and Na⁺ binding. To describe pre-equilibrium kinetics of substrate binding, we monitored fluorescence changes after rapid application of aspartate or Na⁺ to L130W Glt_Ph. Monoexponential fits to the time courses of fluorescence intensities provided time constants in the range of 1s at 22°C. Time constants do not depend on added [aspartate], indicating that aspartate binding is not-rate limiting. In contrast, they decrease with increasing added [Na⁺], suggesting that the rate limiting step is one of the Na⁺-binding events, and kinetic analysis of fluorescence changes can provide information about association and dissociation rate constants. So far, 2 Na⁺ binding sites have been defined by crystallographic analysis (Boudker et al. (2007) Nature 445 (7126), 387–393) in Glt_Ph, whereas two other were proposed based on mutagenesis and functional studies and assigned to residues homologous to D312 and D390 (Tao et al. (2006) J Biol Chem 281, 10263–10272; Tao & Grewer (2007) J Gen Physiol 129, 331–344)). Na⁺ and aspartate are thought to bind in a certain order to the transporter, however, the assertion of these sites to the binding order is not clear. We first focused on the role of D390. In presence of 100mM aspartate, Na⁺ affinity of D390N Glt_Ph was reduced by a factor of 20 compared to WT, supporting the role of D390 in Na⁺ binding. WT transporters displayed Na⁺-dependent fluorescence changes also in the absence of aspartate. D390N abolished Na⁺-dependent fluorescent changes under these conditions, suggesting that D390 is important for Na⁺-binding events that precede aspartate binding. Combining site-directed mutagenesis with fluorescence measurements will allow assigning the binding order of Na⁺ to EAAT-type glutamate transporters and quantification of individual binding steps.