The two-dimensional crystallization of proteins on floating lipid layers has been documented extensively. However, the transfer of such organized protein arrays to solid supports for structural analysis or for use as sensing elements in devices can damage the fragile crystalline films. In this work, we investigated several parameters that influence the growth of two-dimensional protein crystals directly on solid substrates. With streptavidin as the model protein, we considered the effects of epitaxial coupling, surface roughness, and substrate order on the two-dimensional crystallization of streptavidin on biotinylated lipids supported on those substrata. The substrates investigated included lipid-coated atomically flat mica sheets, etched mica, and rough glass slides; silanized glass coverslips; and polymer-supported lipid bilayers. Polarized, reflected light fluorescence microscopy and fluorescence recovery after photobleaching results demonstrated that crystallization is coupled strongly to the mobile lipid fraction in the biotinylated lipid monolayer. The absence of observable crystals on polymer-supported lipid bilayers suggests that, despite their uniform appearance, the latter are likely rough and irregular on a submicroscopic length scale.