The developmental mechanisms involved in the formation of stable arrays of postsynaptic neurotransmitter receptors near sites of neurotransmitter release are essentially unknown. However, several recent studies have shown that cells or tissues of neural origin produce macromolecular factors which induce an increase in the number of acetylcholine (ACh) receptors or the number of receptor aggregates on cultured embryonic myotubes. We have tested primary cultures of embryonic neurons and other cell types for the release of an ACh receptor aggregation factor. Conditioned medium from the cultures was applied to cultures of embryonic rat myotubes for 1 day; ACh receptors on the myotubes were stained with tetramethylrhodamine-labeled alpha- bungarotoxin and ACh receptor aggregation activity, defined as the change in the number of receptor aggregates per myotube, was assayed. Aggregation activity with a molecular weight greater than or equal to 50,000 was released by cultures of neurons from sympathetic ganglia, dorsal root ganglia, spinal cord, and cerebellum. Little or no activity was released by glial or other non-neuronal cultures. Release of aggregation activity by different neuronal cell types varied by up to an order of magnitude; however, this variation was not well correlated with the differences in ACh synthesis. The factor(s) in neuronal cell conditioned medium induced a rearrangement of pre-existing receptors at the cell surface, and its action was not dependent on new protein synthesis. The results of this study are consistent with the idea that one or more receptor aggregation factors secreted by neurons are involved in the organization of neurotransmitter receptors during synapse formation in vivo.