Synapses form between cells of a neuroblastoma X glioma hybrid clone and cultured mouse skeletal myotubes. The synapses are cholinergic, and the acetylcholine release mechanism is dependent on calcium ions. The transmitter output of the synapses is low, with considerable variability in the latency and amplitude of the postsynaptic responses to presynaptic action potentials. The fine structure of physiologically identified functional junctions was examined electron microscopically. Small (50 nm) clear vesicles were seen presynaptically and there were areas with a wide (approx. 50 nm) gap containing basement membrane-like material between the pre- and postsynaptic cells. In addition, in some regions there was a densely staining material lining the muscle membrane and some suggestion of infolding of the muscle membrane. In none of the cases, however, have areas been found where small, clear vesicles cluster around pre- and postsynaptic membrane densities. Thus, functional synapses can occur in the absence of the highly organized synaptic structure seen at mature synapses.