Excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft to ensure low resting glutamate concentrations and to terminate glutamatergic synaptic transmission. EAATs are not only secondary-active glutamate transporters, but also anion-selective channels. Whereas glial glutamate transporters (EAAT1 and 2) are crucial for glutamate clearance after release, neuronal isoforms (EAAT3, 4 and 5) might also regulate neuronal excitability through their associated anion conductance. We here focus on anion conduction by a neuronal EAAT isoform, EAAT4. EAAT4 anion channels display voltage-dependent gating that is modified by glutamate and permeant anions. We expressed EAAT4 in mammalian cells, and studied gating of EAAT4 anion channels through whole-cell patch clamping. In the absence of glutamate, WT EAAT4 anion currents are activated by membrane hyperpolarisation. Application of glutamate inverses the voltage dependence of gating resulting in time-dependent increases of anion currents at positive potentials. In the absence of glutamate, gating of EAAT4 anion channels is not affected by the external anion composition and only slightly by the internal anions. However, the application of glutamate makes the WT EAAT4 anion channels sensitive to changes of external and internal anions. Increasing the external anion concentration shifts the activation curve of EAAT4 to more negative potentials (DV_{1/2(1®140 mM NO3)} = -82.5 19.6 mV, (n = 3)). The presence of external anions additionally slows deactivation during hyperpolarization at WT EAAT4 (t_{[1mM NO3]} = 16.5 0.8 ms (n = 5)) and t_{[140mM NO3]} = 37.9 1.2 ms (n = 3)). Increasing internal anion concentrations shifts the voltage-dependence of activation of WT EAAT4-associated anion channels to the right (DV_{1/2(1®40 mM NO3)} = +103.9 4.6 mV, (n = 3)). The highly conserved D112 at the end of TM2 was recently shown to be a determinant of EAAT1-mediated anion currents (Ryan et al., (2004) J. Biol. Chem., 279, p. 20742-20751). We studied the effects of extra- and intracellular anions on the corresponding mutant (D117A) in EAAT4. The D117A EAAT4-mediated anion currents are strictly outward rectifying current, and are not modified by changes of [glutamate] and [anions]. We conclude that anions and glutamate modify EAAT anion channel gating in a distinct way and that TM2 is a molecular determinant of this regulation.