Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) smooth muscles. Conductances, such as Ca2+-activated Cl− channels (CaCC) and non-selective cation channels (NSCC) have been proposed to mediate pacemaker activity. We characterized the expression of CaCC in gastrointestinal (GI) muscles. Anoctamin 1 (ANO1), encoded by Tmem16a) was originally found to be highly expressed in ICC in a microarray screen. Several splice variants of Tmem16a are expressed in GI muscles, as well as other paralogues of the Tmem16 family. ANO1 protein is abundant and specifically distributed in all classes of ICC in murine, non-human primate (Macaca fascicularis) and human GI tracts. The effects of CaCC blocking drugs, niflumic acid and 4,4′-diisothiocyano-2,2′-stillbene-disulfonic acid (DIDS) were tested on spontaneous slow wave activity in intact muscles of mouse, primate, human small intestine and stomach. Block of CaCC blocked slow waves in a concentration-dependent manner. The mechanism of pacemaker activity was investigated in cells isolated from transgenic mice with constitutive expression of a green fluorescent protein (copGFP) in ICC. Depolarization of ICC caused large amplitude inward currents that were due to a Cl− selective conductance as evaluated by reversal of tail current analysis. Removal of extracellular Ca2+, replacement of Ca2+ with Ba2+, or extracellular Ni2+ (30 μm) blocked the inward current. Single Ca2+-activated Cl− channels with a unitary conductance of 7.8 pS were resolved in excised patches from ICC. Properties of the single channels were similar to ANO1 channels (8 pS) expressed in HEK293 cells. The inward current was blocked in a concentration-dependent manner by niflumic acid (IC50= 4.8 μm). Under current clamp conditions, transient depolarizations occurred spontaneously, and these events were blocked by niflumic acid. In intact muscles slow waves failed to develop in mice with Tmem16a deactivated (Tmem16atm1Bdh/tm1Bdh). This was not a delayed developmental error as pacemaker activity did not develop many days after birth. Absence of ANO1 and electrical pacemaker activity did not inhibit the development of ICC networks, because normal appearing cells were abundant. These data demonstrate that ICC have a prominent CaCC that contributes to the generation of pacemaker activity.