1. Interstitial cells of Cajal (ICC) have been shown to generate pacemaker activity in gastrointestinal (GI) muscles. Experiments were performed to characterize the ICC within the canine gastric antrum and to determine the site(s) of pacemaker activity and whether active propagation pathways exist within the thick-walled tunica muscularis of large mammals. 2. Immunohistochemistry and electron microscopy revealed four populations of ICC within the antral muscularis on the basis of anatomical location. Typical ICC were found in the myenteric region of the small intestine (IC-MY). Intramuscular ICC (IC-IM) were intermingled between muscle fibres of circular and longitudinal muscle layers. ICC were also found within septa (IC-SEP) between muscle bundles and along the submucosal surface of the circular muscle layer (IC-SM). ICC were identified in each location by ultrastructural features. 3. Intracellular electrical recordings demonstrated nifedipine-insensitive slow waves throughout the circular muscle layer. Separation of interior and submucosal circular muscle strips from the dominant (myenteric) pacemaker region dramatically slowed frequency but did not block spontaneous slow waves, suggesting that pacemaker cells populate all regions of the circular muscle. 4. Slow waves could be evoked in interior and submucosal circular muscles at rates above normal antral frequency by electrical pacing or by acetylcholine (0.3 microM). Active slow wave propagation occurred in all regions of the circular muscle, and propagation velocities were similar in each region. 5. In summary, antral muscles of the canine stomach have pacemaker capability throughout the circular muscle. Normally, a dominant pacemaker near the myenteric plexus drives slow waves that actively propagate throughout the circular layer. Pacemaker activity and the active propagation pathway may occur in networks of ICC that are distributed in the region of the myenteric plexus and throughout the circular muscle layer.