The synapse-organizing protein agrin is expressed by muscle cells. In this study we begin to characterize the role of muscle agrin in synapse formation by investigating its distribution and expression during the formation of nerve-induced ACh receptor (AChR) aggregates in vitro. We have used species-specific anti-agrin antibodies to show that muscle agrin colocalizes with nerve-induced AChR clusters in chimeric nerve- muscle cocultures. Furthermore, quantitation by radioimmune assay shows that the expression of muscle agrin by cultured chick myotubes is increased more than twofold by coculture with cells from the motoneuron- rich ventral spinal cord. These data suggest a role for muscle agrin in neuromuscular junction development.