Background The intravenous anaesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, we explored the influence of the large intracellular loop (LIL) in propofol sensitivity of both receptors. Methods We screened the LIL of α1 GlyRs and α1β2 GABAARs using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with WT (wild type) or mutant receptors. Results Alanine mutation of a conserved phenylalanine residue within the α1 LIL significantly reduced propofol enhancement in both GlyRs (360±30 vs 75±10%, mean±SEM) and GABAARs (361±49% vs 80±23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol and isoflurane. At the single channel level, the ability of propofol to increase open probability was significantly reduced in both α1 GlyR (189±36 vs 22±13%) and α1β2 GABAAR (279±29 vs 29±11%) mutant receptors. Conclusion In this study, we demonstrate that the LIL of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Our results suggest a new role of the LIL in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol.