Dissociated neuronal cultures combined with planar-type microelectrode arrays (MEAs) have been used as a promising read-out platform for the application of cell-based biosensors. There are increasing interests in engineering neuronal cultures to form the desired network topology by surface micropatterning technology. Here, we report a long-term cultivation of primary hippocampal neurons on microelectrode arrays using soft-lithography. Ordered hippocampal neuronal networks were formed by seeding neurons in agarose-microwells and inducing neurite outgrowth through microgrooves. Unlike previous approaches, our technique allowed us to design networks with various microwells on microelectrode arrays with high repeatability. These hippocampal network chips were cultivated for 30 days with excellent pattern fidelity, and neural spikes were successfully measured. We also found that spontaneous activity of the networks could be enhanced by acute disinhibition of inhibitory synapses. The proposed patterning method for neuronal network chips will be a potentially powerful tool for cell-based drug-screening applications.