Rat superior cervical ganglia (SCG), which are sympathetic ganglia, neurons and ventricular myocytes (VMs) were co-cultured separately in a minichamber placed on a microelectrode-array (MEA) substrate. The minichamber was fabricated photolithographically and had 2 compartments, 16 microcompartments and 8 microconduits. The SCG neurons were seeded into one of the compartments and all of the microcompartments using a glass pipette controlled by a micromanipulator and a microinjector. The VMs were seeded into the other compartment. Three days after seeding of the VMs, the neurites of the SCG neurons had connected with the VMs via the microconduits. Electrical stimulations, trains of biphasic square pulses, were applied to the SCG neurons in the microcompartments using 16 electrodes. Evoked responses were observed in several electrodes while electrical stimulation was applied to the SCG neurons. According to the two-way analysis of variance (ANOVA), the beat rate after electrical stimulation was affected by the frequency and the number of the stimulation pulses. These results suggest that pulse number and the frequency of the electrical stimulation contribute to modulation of the beat rate of the cardiomyocytes.