1. The influence of target interaction upon the electrophysiological properties of dissociated ciliary ganglion cells was investigated by testing the sensitivity of the neuronal somal membrane to ionophoretically applied acetylcholine (ACh). Variations in the percentage of cells responsive to the transmitter were measured with time in culture. 2. Twenty‐four hours after plating, all cells respond to an ionophoretic pulse of ACh with a depolarization. However, 1 week after plating (between 7 and 14 days) most of the neurones are unresponsive, and highly responsive cells (greater than 100 mV peak depolarization/nC) are extremely rare. At even later times in culture, neurones sensitive to the transmitter are again more frequent. 3. When neurones are plated onto pre‐formed pectoral myotubes, however, ACh sensitivity is maintained throughout a 3 week culture period. Neuromuscular junctions are formed by the neurones, and when sufficient neurones are present, all the muscle fibres tested show evidence of functional synaptic transmission. Chemosensitivity to ACh is not maintained by neurones in muscle‐free microcultures are present on the same cover‐slip. 4. Interneuronal synaptic contacts, defined by ultrastructural criteria, are formed in cultures of neurones alone, but evidence of widespread functional synaptic interaction between cells was not found at 7‐14 days in culture. 5. It is concluded that the maintenance of ACh sensitivity of cultured ciliary ganglion cells is enhanced by the presence of muscle in co‐culture. The interneuronal synaptic contacts observed are apparently not as potent a stimulus as co‐culture with muscle for the full expression of the cholinergic phenotype under these culture conditions.