Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized 'slow wave' current. Reversal of tail current analysis showed this current was due to a Cl(-) selective conductance. ICC express ANO1, a Ca(2+)-activated Cl(-) channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca(2+), replacement of Ca(2+) with Ba(2+), or extracellular Ni(2+) (30 microm) blocked the slow wave current. Single Ca(2+)-activated Cl() channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC(50) = 4.8 microm). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca(2+)-activated Cl(-) conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.