AChR is concentrated at the postjunctional membrane at the neuromuscular junction. However, the underlying mechanism is unclear. We show that α-actinin, a protein known to cross-link F-actin, interacts with rapsyn, a scaffold protein essential for neuromuscular junction formation. α-Actinin, rapsyn, and surface AChR form a ternary complex. Moreover, the rapsyn-α-actinin interaction is increased by agrin, a factor known to stimulate AChR clustering. Downregulation of α-actinin expression inhibits agrin-mediated AChR clustering. Furthermore, the rapsyn-α-actinin interaction can be disrupted by inhibiting Abl and by cholinergic stimulation. Together these results indicate a role for α-actinin in AChR clustering.