Endocytosis involves the capture of membrane from the cell surface in the form of vesicles, which become rapidly acidified to about pH 5. Here we show using atomic force microscopy (AFM) imaging that this degree of acidification triggers phase separation in lipid bilayers containing mixed acyl chains (e.g. palmitoylioleoyl) or complex mixtures (e.g. total brain extract) but not in bilayers containing only lipids with unsaturated chains (e.g. dioleoyl). Since mixed-chain lipids are major constituents of the outer leaflet of the plasma membrane, the type of phase separation reported here might support protein clustering and signaling during endocytosis. (C) 2010 Elsevier Inc. All rights reserved.