The interactions of cationic lipopeptide C15H31CO-Trp-Lys-D-Leu-Lys with model lipid bilayers supported on gold surface were investigated using electrochemical methods combined with atomic force microscopy imaging and quartz crystal microbalance measurements. It has been found that the mode of the lipopeptide action strongly depends on the net charge of the lipid membrane. In case of the negatively charged bilayers composed of L-alpha-phosphatidylethanolamines and L-alpha-phosphatidylglycerols extracted from E. coli bacteria, C15H31CO-Trp-Lys-D-Leu-Lys molecules initially aggregate on the top of the membrane as a result of the electrostatic attraction between cationic peptide moiety and anionic polar heads of phosphatidylglycerols. Further fusion of the aggregates leads to the swelling and partial disruption of the membrane, while its permeability is substantially increased. Interestingly, the changes in the molecular organization of negatively charged membrane were much more pronounced in the upper leaflet of the bilayer. The effect of C15H31CO-Trp-Lys-D-Leu-Lys on zwitterionic bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol was definitely different. In this case, the disturbance of the membrane structure was rather subtle. It has been observed that a small fraction of lipopeptide molecules rapidly inserts into the bilayer and perturbation of the assembly is homogenous throughout the film. This was followed by a noticeable decrease in membrane permeability and stiffening of the lipid bilayer. Such sealing effect indicates that the insertion of lipopeptide did not result either in pore formation or membrane rupture. (C) 2018 Elsevier Ltd. All rights reserved.