Electrochemical scanning tunneling microscopy (EC-STM) has been applied to study the structure of the film formed by fusion of cholesterol suspensions and mixed dimyristoylphosphatidylcholine (DMPC)/cholesterol vesicles on a Au(111) electrode surface. It has been demonstrated that cholesterol molecules assemble at the gold support into several structures templated by the crystallography of the metal surface and involving flat or edge-on adsorbed molecules. Studies of the film formed by fusion of mixed DMPC/cholesterol vesicles revealed that ordered domains of either pure DMPC or pure cholesterol were formed. These results indicate that, at the metal surface, the molecules released by the rupture of a vesicle initially self-assemble into a well-ordered monolayer. The self-assembly is controlled by the hydrocarbon skeleton-metal surface interaction. In the case of mixed DMPC/cholesterol vesicles, the molecule-metal interactions induce segregation of the two components into single component domains. However, the molecule-metal interaction induced monolayer is a transient phenomenon. When more molecules accumulate at the surface, the molecule-molecule interactions dominate the assembly, and the monolayer is transformed into a bilayer.