Supported lipid bilayers (SLBS) and two-dimensional protein assemblies formed on solid supports of various roughnesses were characterized by atomic force microscopy (AFM). The presence of SLBs was detected reliably by force measurements and by imaging. Three types of responses could be distinguished depending on the applied loads. These responses are interpreted as due to transient restructuration of the lipid assembly in the region of contact between lipid-covered support and AFM tip, driven by hydrophobic/hydrophilic interactions. Two-dimensional crystals of streptavidin could be resolved on SLBs formed on silicon wafers, whereas annexin A5, previously shown to crystallize on mica-SLBs, formed a close-packed noncrystalline assembly on lipid bilayers supported by silicon wafers.