Interstitial cells of Cajal (ICC) have been suggested as pacemaker cells in the gastrointestinal tract. A method was developed to isolate ICC from the slow-wave pacemaker region of the canine proximal colon. These cells were identified under phase-contrast microscopy, and their identity was verified by comparing their ultrastructure with the morphology of ICC in situ. Patch-clamp experiments demonstrated that these cells are excitable; voltage-dependent inward and outward currents were elicited by depolarization. Inward current transients were identified as calcium currents. A portion of the outward current appears to be due to Ca2+-activated K channels commonly expressed in these cells. ICC were also spontaneously active, generating electrical depolarizations similar in waveform to slow-wave events of intact colonic muscles. These findings are consistent with the hypothesis that ICC initiate rhythmicity in the colon.