Rat superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) were co-cultured in chambers made of polydimethylsyloxane. The chambers were placed on a microelectrode-array (MEA) substrate and connected with a pathway. 24 hours after dissemination of the VMs, neuntes of the SCG neurons outgrew through the pathway and reached the VMs. Spontaneous electrical activities of the SCG neurons and the VMs were observed several days after the dissemination. Constant-voltage stimualtion (1 V, 1 ms, biphasic square pulses) was applied to the SCG neurons at the frequency of 10 Hz using 32 electrodes. Contraction rate of the VMs increased by 153 +/-110 % immediately after the stimulation to the SCG neurons was stopped. Then contraction rate gradually decreased and returned to almost the same rate as before the stimulation 5 minutes after the 1-min stimulation. Propranolol (beta-adrenergic receptor antagonist) prevented contraction rate of the VMs from increasing after electrical stimulation to the SCG neurons. These results suggest that neuromuscular junctions were formed between the SCG neurons and the VMs. Overall the semi-separated co-culture system in this study is available in research on changes in contraction rate of the VMs after applying electrical stimulation to the SCG neurons.