The concentration of glutamate within a glutamatergic synapse is tightly regulated by the excitatory amino acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl- conductance. This Cl- conductance is activated by the binding of substrate and Na+, and the direction of Cl- flux is independent of the rate or direction of substrate transport, and thus the two processes are thermodynamically uncoupled. This Cl- conductance is well conserved amongst the glutamate transporter family, and has been proposed to play roles in regulating ionic homeostasis and glutamate release in the retina. Several crystal structures of an EAAT archaeal homologue, GltPh, at different stages of the transport cycle have been solved. In a recent structure, a small cavity at the interface of the transport and trimerisation domains has been identified. This cavity is primarily lined by polarisable residues and contains several residues that have been implicated in Cl− permeation. We hypothesise that throughout the transport cycle this cavity opens up to form the Cl− channel. In this study, mutagenesis of residues which line this cavity in EAAT1, and two-electrode-voltage-clamp electrophysiology have been utilized to determine if this cavity forms part of the Cl− permeation pathway. Additionally, double cysteine mutants were generated in a cys-less EAAT1 background and analysed in an attempt to trap the protein in a purely Cl− conducting state. Significant alterations in Cl- conductance properties were observed when Ser366, Leu369, Phe373, Arg388, Pro392, and Thr396 were mutated to small hydrophobic residues. Additionally, cys-less EAAT1 double cysteine mutants K300C/L367C and M89C/I469C demonstrated the ability to crosslink and trap EAAT1 in a Cl- conducting state. Together, these results suggest that the EAAT1 Cl- permeation pathway is located at the interface of the transport and trimerisation domains.