The property of the dyes, acridine orange and methylene blue, to exhibit metachromatic changes upon binding to negatively charged groups that are within a defined spatial separation was employed to study the lateral and transverse topography of sulfatide and gangliosides GMI and GDt a mixed with dipalmitoylphosphatidylcholine (DPPC) in unilamellar vesicles. The spectral changes of the dyes in the presence of liposomes containing anionic glycosphingolipids (GSLs) (hypochromism and frequency shift) are typical of polyanionic lattices while minor changes are found for neutral lipids. The metachromatic changes are abofisbed by the presence of Ca 2+ in the external medium. The proportion of anionic GSLs accessible to the dyes on the external surface of the liposomes is greater as the GSLs are more complex (sulfatide < GMt < GDla) and as its proportion in the mixture decreases. The number of molecules of anionic GSLs that are laterally distributed on the external surface in a position favorable for the formation of dye dimers (at intermolecular distances not exceeding 1 nm) is greater for snifatide than for 8anglioside. This is correlated to the greater intermolecniar distances and delocalization in ganglioside-, compared to sulfatidecontaining interfaces. The experimental values indicate that the mixture with DPPC of any of the anionic GSLs studied behaves as if it was more enriched in the GSLs compared to the proportions of the whole mixture.