Agrin, which induces acetylcholine receptor (AChR) clustering at the developing neuromuscular synapse, occurs in multiple forms generated by alternative splicing. Some of these isoforms are specific to the nervous system; others are expressed in both neural and nonneural tissues, including muscle. We have compared the AChR clustering activity of agrin forms varying at each of the three identified splicing sites, denoted x, y, and z. Agrin isoforms were assayed by applying either transfected COS cells, with agrin bound to their surfaces, or soluble agrin to myotubes of the C2 muscle line, or of two variant lines having defective proteoglycans. Dramatic differences in activity were seen between z site isoforms and lesser differences between y site isoforms. The most active agrin forms contained splicing inserts of 4 amino acids at the y site and 8 amino acids at the z site. These forms are found exclusively in neural tissue. All forms were active on C2 myotubes in cell-attached assays, but muscle forms were less active than neural forms. AChR clustering activity of all agrin forms was decreased when assayed on the proteoglycan-deficient lines, suggesting that proteoglycans may help mediate the action of agrin. As neural agrin forms are more active than muscle forms, they are likely to play a primary role in synaptogenesis.