The mechanism of membrane permeabilization by the antifungal heptaene macrolide amphotericin B (AmB) was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). The disruptive permeabilization of a sterol-free mercury-supported lipid monolayer by low AmB concentrations, after a sufficiently long exposure to the macrolide, suggests a similar effect on the distal lipid monolayer of vesicular or bilayer lipid membranes. The notable permeabilization of a tBLM to Cd(II), with amalgam formation, by 0.6 mu M AmB complexed with sterols in a 1:3 molar ratio, as compared with the absence of any effect in the absence of sterols, suggests a direct participation of sterols in ion channel formation. The fact that the tBLM permeabilization requires the presence in solution of a freshly added AmB-sterol mixture and of Cd(II) or Ca2+ ions points out the notable role played by these divalent inorganic ions in the formation of AmB ion channels. Ion channels formed in a tBLM by AmB-ergosterol complexes are stable at physiological transmembrane potentials, whereas those formed by AmB-cholesterol complexes require application of non-physiological transmembrane potentials for their stability. (C) 2013 Elsevier Ltd. All rights reserved.