Aneural clustering of the acetylcholine receptor (AChR) is an early post_synaptic step of neuromuscular junction assembly. We investigated the role of basement membrane components in this process in C2C12 myotubes. Induction of clustering by laminins_111 and_211 was found to be substantially dependent upon their interaction with nidogen_1 since ablation of the nidogen_binding site by an N802S point mutation in the ã1_chain prevented clustering. The laminin_nidogen complex activity, dependent also on laminin polymerization, could be mimicked by incubation of laminin_211ã1N802S with nidogen_antibody crosslinking of nidogen_1 on the myotube surface. Deletion of laminin LG4_5 in heterotrimeric laminin, in contrast to deletion of LG1_3, also prevented AChR clustering, implicating the terminal LG repeats in the clustering process. Combined antibody ligation of isolated LG4_5 and nidogen_1 mimicked the clustering observed with laminin_nidogen, as did antibody ligation of α_dystroglycan and β1_integrin. Collectively the data was compatible with a binary mechanism whereby laminin ligates dystroglycan and aggregates nidogen that in turn ligates β1_integrin. The role laminin_nidogen interaction was also investigated in developing diaphragm muscle of transgenic mice that lacked the nidogen_binding LE repeat in laminin ã1 as described in Willem et al., 2002. The size and distribution of AChR clusters was found to be altered at E14.5 consistent with the findings on cultured myotubes, supporting the suspected role of the laminin_nidogen complex in vivo.