Total internal reflection fluorescence microscopy (TIRFM) has been utilized to explore the effect of cationic protein lysozyme (Lz) on the morphology of solid-supported lipid bilayers (SLBs) comprised of zwitterionic lipid phosphatidylcholine (PC) and its mixture with anionic lipid cardiolipin (CL). Kinetic TIRFM imaging of different systems revealed subtle interplay between lipid lateral segregation accompanied by exchange of neutral and acidic lipids in the protein-lipid interaction zone, and the formation of lipid multilayer stacks. The switch between these states was shown to be controlled by CL content. In weakly charged SLBs containing 5. mol% CL, assembling of CL molecules into planar domains upon Lz adsorption has been observed while at higher content of anionic lipid (25. mol%) in-plane domains tend to transform into multilayer stacks, thereby ensuring the most thermodynamically-favorable membrane conformation. © 2010 Elsevier B.V.