Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre-and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes. (C) 2009 Elsevier Inc. All rights reserved.