Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.