The deposition of amyloid beta-protein (A beta) is a pathological hallmark of Alzheimer's disease (AD). We previously found that the ganglioside-enriched microdomains (ganglioside clusters) in presynaptic neuronal membranes play a key role in the initiation of the A beta assembly process. However, not all ganglioside clusters accelerate A beta assembly. In the present study, we directly observed a spherical A beta in an atomic force microscopic study on the morphology of a reconstituted lipid bilayer composed of lipids that were extracted from a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from aged mouse brain. The A beta assembly was generated on a distinctive GM1 domain, which was characterized as the A beta-sensitive ganglioside nanocluster (ASIGN). By using an artificial GM1 cluster-binding peptide, ASIGN was found to have a high density of GM!; therefore, there would be a critical density of GM1 in nanoclusters to induce A beta binding and assembly. These results suggest that ganglioside-bound A beta (GA beta), which acts as an endogenous seed for A beta fibril formation in AD brains, is generated on ASIGN on synaptosomal membranes.