Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.