||At neuromuscular junctions, there is a local increase in the synthesis and aggregation of acetylcholine receptors (AChRs) on the muscle fiber. Agrin, a protein isolated from the synaptic basal lamina, is proposed to induce the aggregation of AChRs. Neuregulin, originally purified from chick brain, is proposed to regulate the synthesis of AChR in the muscle fiber. Both motor neuron and muscle express agrin and neuregulin in vivo. An in vitro NG108-15 cell-muscle coculture model was used to elucidate the roles of agrin and neuregulin in the formation of neuromuscular junctions. NGlOB-15 cells, a neuroblastoma x glioma hybrid cell line, have been shown to form synapses with cultured myotubes, and induce the synthesis and aggregation of AChRs when cocultured with myotubes. Several lines of evidence indicate that NGl08- 15 cells express both agrin and neuregulin in culture. The expression of agrin and neuregulin, in either neurons or myotubes, could be blocked by antisense cDNA transfection. The agrin-deficient NGlOS-15 cells lost their AChR-aggregating activity, whereas the aggregation of AChRs could be induced in agrin-deficient myotubes when they were challenged with agrin or neurons. Similarly, the expression of neuregulin was inhibited in NG108-15 cells by the antisense neuregulin cDNA transfection, while the AChR-inducing activity of NG108-15 cells was reduced. These results demonstrate that the neuron-derived agrin and neuregulin are the primary factors in directing the formation of neuromuscular junctions.