Amphotericin B UV-Vis absorption linear dichroism data was combined with Nystatin steady-state fluorescence linear dichroism data to calculate their orientational probability density functions in multilayers of dipalmitoylphosphatidylcholine (DPPC), DPPC+cholesterol and arachidic acid+cholesterol multilayers. The cholesterol effect is striking on both the mean orientation and range of allowed orientations. The mean becomes closer to the layers normal (from 8.6degrees to 2.3degrees) in DPPC multilayers when cholesterol is present and the range (i.e., full distribution width) narrows from congruent to40degrees to congruent to10degrees. However, a small fraction of the antibiotic eventually remains parallel to the multilayer surface, possibly adsorbed to it. The results favor the pore model for the biochemical mode of action of the antibiotics. Moreover, a direct involvement of the sterols is suggested.