GABA receptors (GABAARs) mediate inhibition in the adult brain. These channels are heteropentamers and their ligand binding sites are localized at the β+ / α- interfaces. As expected, mutations of binding-site residues affect binding kinetics but accumulating evidence indicates that gating is also altered, although the underlying mechanisms are unclear. We investigated the impact of the hydrophobic box residue localized at α1(-), F64 (α1F64), on the binding and gating of rat recombinant α1β1γ2 receptors. The analysis of current responses to rapid agonist applications confirmed a marked effect of α1F64 mutations on agonist binding and revealed surprisingly strong effects on gating, including the disappearance of rapid desensitization, the slowing of current onset, and accelerated deactivation. Moreover, nonstationary variance analysis revealed that the α1F64C mutation dramatically reduced the maximum open probability without altering channel conductance. Interestingly, for wild-type receptors, responses to saturating concentration of a partial agonist, P4S, showed no rapid desensitization, similar to GABA-evoked responses mediated by α1F64C mutants. For the α1F64L mutation, the application of the high-affinity agonist muscimol partially rescued rapid desensitization compared with responses evoked by GABA. These findings suggest that α1F64 mutations do not disrupt desensitization mechanisms but rather affect other gating features that obscure it. Model simulations indicated that all of our observations related to α1F64 mutations could be properly reproduced by altering the flipped state transitions that occurred after agonist binding but preceded opening. In conclusion, we propose that the α1F64 residue may participate in linking binding and gating by influencing flipping kinetics.