The supramolecular structure of Niosomal vesicles (Niosomes) made of a binary mixture of polysorbate 20 ( Tween 20) and Cholesterol in aqueous solution was investigated by means of synchrotron small angle X-ray scattering (SAXS). Solid-supported Niosomal membranes at full hydration exhibit the same structural properties, as determined by in situ energy dispersive X-ray diffraction (EDXD), than their counterpart in solution. Both Niosomes and solid-supported Niosomal membranes are made of highly swollen bilayers rich in Tween 20 coexisting with Cholesterol crystallites. EDXD patterns from oriented samples suggest that at least some Cholesterol crystals are aligned along the normal to the solid support. (C) 2008 Elsevier B.V. All rights reserved.