Lectins such as VVA-B4, which bind N-acetylgalactosaminyl (GalNAc)-terminated saccharides, selectively stain the neuromuscular junction, thus defining a synapse-specific carbohydrate. In seeking roles for this carbohydrate, we asked whether VVA-B4 affected the distribution of acetylcholine receptors (AChRs) on cultured muscle cells. We found that incubation of myotubes with VVA-B4 induced formation of AChR clusters and potentiated the effect of a nerve-derived clustering factor, agrin. Additional experiments implicated GalNAc-terminated glycoconjugates as modulators of agrin-induced AChR clustering. Enzymatic removal of GalNAc residues or treatment with a multivalent protein-GalNAc conjugate blocked agrin-induced clustering, whereas enzymatic unmasking of additional GalNAc residues induced clustering in the absence of added agrin. Moreover, incubation with agrin led to redistribution of VVA-B4-binding material on myotubes. Together, these results suggest that agrin-induced clustering of AChRs involves a GalNAc-dependent step.