The bacterial mechanosensitive channel MscS plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. MscS functional characteristics have extensively been studied in both giant spheroplasts and liposomes. Despite many studies of MscS reconstituted into liposomes the channel orientation in liposomal membranes is still unknown. We examined the orientation of MscS in liposomes by patch-clamp and confocal microscopy. using its previously determined electrophysiological and pharmacological properties we were able to determine that in liposomes MscS retains the right-side-out orientation as in giant spheroplasts based on the following evidence: (i) I-V curves recorded in both spheroplast and liposome preparations exhibited strong outward rectification at both negative and positive pipette pressures. (ii) MscS activation ratio in liposome patches at positive relative to negative pipette voltages and vice versa showed positive correlation at both positive and negative pipette pressures similar to MscS in inside-out excised spheroplast patches. (iii) MscS exhibited a voltage-dependent hysteresis upon application of saw-tooth pressure ramps in both spheroplasts and liposomes. In both spheroplasts and liposomes the hysteresis was more pronounced upon positive pipette voltages compared to negative voltages. (iv) 2.5% of 2,2,2-trifluoroethanol (TFE) caused MscS inactivation in liposome patches when added to the cytoplasmic side of MscS, whereas addition of TFE to the periplasmic side did not inactivate the channel, although it caused a shift of the channel activation towards lower pipette pressures. We obtained a similar result when applying TFE to MscS in spheroplast patches. In conclusion, our findings strongly indicate that the cytoplasmic domain of MscS in liposome membrane patches faces the bath solution as in spheroplast patches. Consequently, upon liposome reconstitution MscS channels preserve their right-side-out orientation comparable to what was previously reported for the MscL channels.Supported by the NH & MRC.