Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission and maintain extracellular glutamate concentrations in the central nervous system below excitotoxic levels. EAAT3 and EAAT4 are two neuronal glutamate transporters that are functionally very different. To further characterize these differences, we studied transporter- associated anion channels using heterologous expression in mammalian cells and oocytes and macroscopic current recordings. Both EAAT-associated anion channels display a SCN>NO3>Cl conductivity and permeability sequence. Glutamate modifies permeation and gating, and the glutamate dependences of the anion current amplitude depend on the anion distribution across the membrane. When cells are dialyzed with a SCN--based solution and externally perfused with a Cl--based solution, EAAT3 and EAAT4 show similar voltage-dependent gating, in the absence as well as in the presence of glutamate. In contrast, at the inverted anion distribution, EAAT3 anion channels are activated by membrane depolarisation and EAAT4 channels by hyperpolarisation. Increasing glutamate concentrations augment EAAT4 anion currents with a sigmoidal relationship, indicating cooperative interaction between different carrier domains. This interaction is absent in EAAT3. Thus, diverse EAAT isoforms differ in the functional properties of associated anion channels and in the interactions between transporter subunits.