The clustering of acetylcholine receptors (AChRs) in the post-synaptic membrane of skeletal muscle is an early developmental event in the formation of the neuromuscular junction. Several studies show that laminin, as well as neural agrin, can induce AChR clustering in C2C12 myofibers. We recently showed that specific isoforms of the alpha7beta1 integrin (a receptor normally found at neuromuscular junctions) colocalize and physically interact with AChR clusters in a laminin-dependent fashion. In contrast, induction with agrin alone fails to promote localization of the integrin with AChR clusters. Together both agrin and laminin enhance the interaction of the integrin with AChRs and their aggregation into clusters. To further understand this mechanism we investigated cluster formation and the association of the alpha7beta1 integrin and AChR over time following induction with laminin and/or agrin. Our results show that the alpha7beta1 integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable association of the integrin and AChRs and this association is independent of clustering. In addition to laminin-1, merosin (laminin-2/4) is present both before and after formation of neuromuscular junctions and also promotes AChR clustering and colocalization with the integrin as well as synergism with agrin. Using site directed mutagenesis we demonstrate that a tyrosine residue in the cytoplasmic domain of both (&agr;)7A and (&agr;)7B chains regulates the localization of the integrin with AChR clusters. We also provide evidence that laminin, through its association with the alpha7beta1 integrin, reduces by 20-fold the concentration of agrin required to promote AChR clustering and accelerates the formation of clusters. Thus laminin, agrin and the alpha7beta1 integrin act in a concerted manner early in the development of the post-synaptic membrane, with laminin priming newly formed myofibers to rapidly and vigorously respond to low concentrations of neural agrin produced by innervating motor neurons.