In this work, we studied the influence of the polyene antibiotic amphotericin B (AmB) on dipalmitoylphosphatidylcholine (DPPC) multibilayers modified by cholesterol and ergosterol investigated by means of X-ray diffraction. The periodic structures related to AmB-DPPC-sterol complexes in multibilayers are completely created in temperatures above the main lipid phase transition. The differences between all observed multilayer structures are related to the thickness. Multibilayers composed of lipid and sterols have similar thickness of 51 angstrom. Addition of amphotericin B leads to creation of new periodic structures. Multibilayers composed of lipid-ergosterol-AmB have thickness of 44 angstrom which does not change with temperature. Multibilayers composed of lipid-cholesterol-AmB at 20 degrees C are 43 angstrom and gets thicker at 50 degrees C. Most probably this is caused by weaker van der Vaals cholesterol-amphotericin B interactions. (C) 2014 Elsevier B.V. All rights reserved.