To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.