Formation of supported lipid bilayers on soft polymer cushions is a useful approach to decouple the membrane from the substrate for applications involving membrane proteins. We prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid, on glass and silicon substrates. (CHI/FIA)(5) films were characterized by atomic force microscopy, giving an average thickness of 57 nm and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and 6.5 on (CHI/HA)(5) films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was attributable to formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.