Aims: Excitatory amino acid transporters (EAATs) mediate two different transport processes, electrogenic coupled movement of glutamate and co-substrates and pore-mediated anion conduction. We compared the sodium dependence of EAAT2- and EAAT4-associated anion currents and investigated neutralization of three candidate Na+-binding sites D399, D471, and D486 in hEAAT2. Methods: WT and mutant EAAT2 and EAAT4 were expressed in mammalian cells and studied through whole-cell patch clamp recordings. EAAT anion currents were increased by intracellular dialysis with KNO3 or NaNO3, and measured at external Cl- -based solutions containing various Na+ and glutamate concentrations. Results: EAAT2 anion channels show a unique Na+ dependence. Whereas EAAT4 anion currents are virtually absent without external Na+, EAAT2 anion currents decrease with increasing [Na+] in the absence of glutamate. In both EAAT2 and 4, application of glutamate results in [Na+]- dependent increases of anion current amplitudes. D399N EAAT2 shows a decreased Na+-affinity in the absence of glutamate and block by glutamate at high extracellular [Na+]. D471N EAAT2 anion channels are only blocked by external Na+ in the presence of internal K+, but not of internal Na+. Glutamate does not modulate D471N EAAT2 anion currents. D486N EAAT2 anion channels are activated by external Na+, but blocked by glutamate. Conclusion: EAAT2 anion channels are active in the absence of Na+. Mutations in D399 modify Na+ binding to the empty EAAT2 transporter. Modulation of EAAT2 anion currents by glutamate requires Na+-association to D471. D486 affects Na+, K+ and glutamate translocation in EAAT2.