The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecular (HB-GAM) in AChR clustering. Here the role of an agrin-HB-GAM interaction in this process was examined using cultured Xenopus muscle cells. Agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.