Electrochemical and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) techniques were employed to study the ion channel properties of gramicidin A (gA) in tethered bilayer lipid membranes (tBLMs) with varying concentrations of gA added to the outer leaflet of the bilayer. The electrochemical impedance spectroscopy (EIS) results demonstrate that gA forms conductive ion channels in tBLM systems even at concentrations as low as 0.5 mol%. The impedance of the membrane depended on the gA concentration where a minimum was observed at 10 mol%. When the amount of gA in the bilayer is increased to a concentration above 10%, the ion channels appear to arrange into a non-conducting state. PM-IRRAS spectra suggest that the gA molecules adopt a tilt angle of similar to 29 +/- 4 degrees in the conductive ion channel state. In the non-conducting state, the average tilt angle of the gA molecules is similar to 52 +/- 3 degrees, which implies that gA peptides assume a random orientation in the tBLM. Crown Copyright (C) 2017 Published by Elsevier Ltd. All rights reserved.