||Motor innervation of the skeletal muscle leads to the clustering of acetylcholine receptor (AChR) in the postsynaptic membrane. It was hypothesized that the establishment of a postsynaptic scaffold 'traps' freely diffusing receptors into forming high-density clusters. In addition, lipid rafts were proposed to serve as signaling platforms during cluster formation, but the methods used in lipid raft studies were debated recently. To further test the diffusion-trap hypothesis and to examine the lipid raft model on living cells, quantum dots (Qdots), brightly fluorescent, non-photobleachable nanoparticles, were used to track the movement of receptors and lipid rafts at the molecular level. Here, we provide first direct evidence on the diffusion-trap hypothesis. We found that although the movement of AChRs at sites of synaptogenic stimulation becomes highly restricted, individual receptors away from these sites continue to exhibit free diffusion, thus suggesting that the clustering process does not involve an active accumulation of receptors but is passive, diffusion-driven. On the contrary to recently published work, we found lipid rafts did not concentrate at AChR clusters and disruption of lipid rafts did not affect the formation of AChR clusters and the diffuse AChRs behaved differently from lipid rafts, thus suggesting that the putative lipid rafts are not directly involved in AChR cluster formation. Crosslinking-induced AChR endocytosis in patients could be mimicked by Qdot staining. Endocytic vesicles could fuse with each other during their linear movements along microtubules in a centripetal manner toward the center of the muscle cell. The fusion process was F-actin-dependent. With time, vesicles were redistributed to the periphery of the cell with unknown function. Our data also suggested that this endocytic process is mediated by clathrin and caveolae might also be involved. These findings delineate precisely the endocytosis of AChRs in muscle cells and highlight the utility of Qdots in understanding intracellular processes.