Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.