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 GM1 and GD1a 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 abolished by the presence of Ca2+ 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 less than GM1 less than GD1a) 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 sulfatide than for ganglioside. This is correlated to the greater intermolecular distances and delocalization in ganglioside-, compared to sulfatide-containing 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.