Several features of the clonal cell line BC3H-1 resemble those of embryonic muscle cells at their early stage of development (Patrick et al.: J Biol Chem 252:2143-2153, 1977). Under normal culture conditions, fully differentiated BC3H-1 cells possess a spindle-shaped, muscle-like morphology with a single nucleus. Like embryonic muscle cell counterparts, they neither express the epsilon subunit nor exhibit the clustered organization of the nicotinic acetylcholine receptor (AChR) characteristic of mature myocytes. Instead, AChRs are evenly distributed upon the sarcolemma upon differentiation. Here we report that BC3H-1 cells can be induced to express AChR clusters by co-culturing the cell line with heterologous retinal neurons, which establish contacts with former cells. Clustering was also triggered by pretreating the culture substratum with several cell adhesion molecules. Polynucleation, a phenomenon observed in mature myotubes in vivo, was also observed in BC3H-1 cells under the two sets of experimental conditions. AChR clustering and polynucleation, both characteristic of the mature postsynaptic region, can thus be induced in BC3H-1 cells by at least two quite distinct pathways: neuronal cell contact and/or cell adhesion molecules. It is suggested that heterologous retinal neurons can elicit only an incomplete differentiation of BC3H-1 cells, failing to concentrate the clusters beneath the neuronal endings, and failing to induce expression of the epsilon subunit, characteristic of the mature AChR.