To reveal the unique intrinsic properties of interstitial cells of Cajal (ICC), morphological and electrophysiological characteristics of isolated ICC from the adult mouse small intestine were investigated and compared with those of smooth muscle cells. All typical ultrastructural features of in situ ICC were evident in isolated ICC throughout the isolation procedure and short-term culture. With the use of the nystatin perforated patch-clamp technique, ICC demonstrated spontaneous voltage oscillations that were not abolished by hyperpolarization nor by L-type calcium channel blockers. This rhythmic activity occurred at room temperature at a frequency of 13.9 ± 11.2 cycles/min, with an amplitude of 13.4 ± 11.2 mV at membrane potentials from −20 to −70 mV. Smooth muscle cells from the same culture only generated voltage-sensitive action potentials above the threshold potential of −35 mV. Hyperpolarization as well as the addition of L-type calcium channel blockers abolished the action potentials. In whole cell voltage-clamp recordings from ICC, a large noninactivating outward current was observed to be activated (5% threshold) at −49.6 mV with a half-activation voltage of −18.7 mV and slope factor of 9.9 mV. In contrast, in smooth muscle cells, smaller outward currents with distinctive transient outward currents were present. In conclusion, the generation of L-type calcium channel blocker-insensitive slow waves in membrane potential is a unique intrinsic property of ICC.