This neutron reflectometry study evaluates the structures resulting from different methods of preparing polymer-cushioned lipid bilayers. Four different techniques to deposit a dimyristoylphosphatidylcholine (DMPC) bilayer onto a polyethylenimine (PEI)-coated quartz substrate were examined: 1) vesicle adsorption onto a previously dried polymer layer; 2) vesicle adsorption onto a bare substrate, followed by polymer adsorption; and 3, 4) Langmuir-Blodgett vertical deposition of a lipid monolayer spread over a polymer-containing subphase to form a polymer-supported lipid monolayer, followed by formation of the outer lipid monolayer by either 3) horizontal deposition of the lipid monolayer or 4) vesicle adsorption. We show that the initial conditions of the polymer layer are a critical factor for the successful formation of our desired structure, i.e., a continuous bilayer atop a hydrated PEI layer. Our desired structure was found for all methods investigated except the horizontal deposition. The interaction forces between these polymer-supported bilayers are investigated in a separate paper (Wong, J. Y., C. K. Park, M. Seitz, and J. Israelachvili. 1999. Biophys. J. 77:1458-1468), which indicate that the presence of the polymer cushion significantly alters the interaction potential. These polymer-supported bilayers could serve as model systems for the study of transmembrane proteins under conditions more closely mimicking real cellular membrane environments.