Sympathetic principal neurons, dissociated from superior cervical ganglia of newborn rats, were plated into cultures containing rat skeletal myotubes formed from previously plated primary myoblasts. Electrophysiological evidence is presented that the neurons developed cholinergic synapses with the myotubes. In addition, the neurons developed cholinergic synapses with each other as previously reported [O'Lague et al. (1974) Proc. Nat. Acad. Sci. USA 71, 3602-3606]. The acetylcholine receptors of myotubes differed from those of the neurons in their sensitivities to curare and hexamethonium, in a manner expected of adult muscle and ganglionic receptors. alpha-Bungarotoxin blocked synaptic transmission from neuron to myotube, but not from neuron to neuron in the same culture.