To explore the electrophysiological properties of the interstitial cells of Cajal (ICCs) and fibroblast-like cells (FLCs), we developed a new preparation by treating the murine small intestine with collagenase. This thin muscle layer preparation contained at least two types of interstitial cells around the enteric nerve bundles, and the cluster of smooth muscle cells displayed a rhythmic contraction. We morphologically identified ICCs and FLCs and conducted patch clamp experiments on each type of cell. The c-kit-positive CD34-negative ICCs showed spontaneous and rhythmic potential fluctuations, and a large transient inward current was evoked by depolarization under voltage clamp conditions. Once the inward current was triggered, it took a regenerative time course and lasted approximately 500 ms. The current was inactivated by continuous depolarization, and by removal of external Ca2+. The application of acetylcholine (ACh) prolonged the duration of spontaneous depolarization as well as the depolarization-induced inward current. This inward current showed a reversal potential of around +3 mV and was considered to be due to non-selective cation channels. The c-kit-negative CD34-positive FLCs showed irregular or regular potential fluctuations, and spontaneous outward current was observed under voltage clamp conditions. This outward current showed a reversal potential of around −80 mV and might be classified as a potassium current. We failed to observe major time- and voltage-dependent currents except the above two currents in the interstitial cells.