Multisite extracellular electrical activity and intracellular calcium were recorded simultaneously. Electrical signals were measured using microelectrode array substrates. A novel cell positioning technique was combined with a method for controlling neurite outgrowth, which allowed cell-electrode contacts to be established easily, thus facilitating the electrical recording. Intracellular calcium was measured optically using the indicator fluo 3. Under low-magnesium conditions, cultured rat cortical neurons showed periodic transients of fluo-3 fluorescence, which were synchronized with the periodic bursting observed electrically. The intervals between bursts could be determined by electrical stimulation through the substrate electrodes. The results suggest that functional synaptic connections are formed in the culture system.