The efficiency and the tight control of neurotransmission require the accumulation of synaptic proteins in discrete domains. In neuromuscular junctions, the main form of acetylcholinesterase (AChE) is a hetero-oligomer in which the catalytic subunits are associated to a specific collagen, ColQ. This structural protein is responsible for the insertion and the accumulation of AChE in the synaptic basal lamina. We have analyzed the time-course of acetylcholinesterase and acetylcholine receptors (AChR) mRNAs during mouse muscle cell differentiation in culture. In parallel, we have visualized the formation of AChE and AChR aggregates. We show that AChR clusters form first which correlates with high gamma-subunit mRNA levels. Then, AChE clusters appear with the onset of contraction and correlate with a dramatic increase in AChE, ColQ1 and ColQ1A mRNA levels in muscle cells. At that stage, AChR gamma-subunit levels drop while the expression level of epsilon-subunits increase. AChE aggregates are organized by a ternary complex, which involves direct interactions between ColQ, perlecan and MuSK.