Experimental evidence suggests that GABA ρ1 receptors are potential therapeutic targets for the treatment of a range of neurological conditions, including anxiety and sleep disorders. Homology modelling of the GABA ρ1 extracellular N-terminal domain has revealed a novel hydrophobic area that extends beyond, but not including the GABA-binding site. Phenylalanine 124 (F124) is predicted to be involved in maintaining the structural integrity of the orthosteric-binding site. We have assessed the activity of a series of GABA ρ1 receptors that incorporate a mutation at F124. Wild-type and mutant human GABA ρ1 subunits were expressed in Xenopus laevis oocytes and AD293 cells, and the pharmacology and kinetic properties of the receptors were measured using electrophysiological analysis. Mutation of F124 had minimal effect on receptor pharmacology. However, the rate of deactivation was significantly increased compared to wild type. This study provides further information about the role of residues within a novel hydrophobic area of the GABA ρ1 receptor. This knowledge can help future studies into the design of potent and subtype-selective ligands with therapeutic value.