The inhibition effect of amiloride on alamethicin ion channels was studied in a model zwitterionic floating bilayer lipid membrane (fBLM). The EIS studies indicated that amiloride prevents the transport of ions through the alamethicin channels leading to an overall increase in membrane resistance. The PM-IRRAS data demonstrated that amiloride has no influence on the secondary structure of alamethicin but restricts the insertion of the peptides into the bilayer and blocks ion transport through preformed alamethicin channels. The effect of amiloride on ion channel formation in the floating bilayer formed by a zwitterionic lipid was compared to those of previous studies involving negatively charged fBLMs and tethered zwitterionic lipid bilayers. The findings from these studies show that the effects of amiloride on ion channel formation strongly depend on the mobility and charge of the membrane lipids.