1. Interstitial cells of Cajal (ICCs) have been identified as pacemaker cells in the gastrointestinal (GI) tracts of vertebrates. We have studied the development of ICCs in pacemaker regions and the onset of electrical rhythmicity in the gastric antrum, small bowel and proximal colon of the mouse. 2. ICCs, as detected by c-Kit immunofluorescence, were found during embryogenesis in regions of the GI tract that eventually become pacemaker areas. Prior to birth, these cells were organized into well-structured networks, and by the end of the embryonic period the morphology of ICC networks in pacemaker regions appeared very similar to that observed in adult animals. 3. Electrical rhythmicity was recorded prior to birth (by E18) in the proximal GI tract (stomach and jejunum), and this activity developed to adult-like behaviour within a week after birth. In the ileum and proximal colon rhythmicity developed after birth, and adult-like characteristics were apparent within the first week. 4. Post-junctional responses of smooth muscles to neural inputs could be recorded at birth, and stimulation of intrinsic nerves often led to oscillatory activity resembling slow waves for up to several minutes following brief stimuli. Nerve stimulation augmented spontaneous activity in the proximal portions of the GI tract and elicited rhythmic activity temporarily in quiescent tissues of the distal GI tract. 5. ICCs and rhythmicity developed in an apparently normal manner in tissues isolated at birth and placed in organ culture. These data suggest that the tunica muscularis provides a suitable microenvironment for the development of ICCs and rhythmicity without the need for extrinsic stimuli. 6. Treatment of small intestinal tissues taken from embryos at E15 with neutralizing c-Kit antibodies abolished ICC development and the organization of ICCs into networks that typically occurs during the late embryonic period. Treatment of muscles taken from newborn animals with c-Kit antibodies blocked postnatal development of ICCs, disrupted already established and functional ICC networks, and rendered muscles electrically quiescent. 7. In summary, ICC networks develop in the pacemaker regions of the murine GI tract before birth. Development and organization of ICCs of the myenteric plexus region into networks precedes the development of electrical rhythmicity. Post-natal development of electrical rhythmicity is mainly characterized by enhancement of the amplitude and frequency of slow waves. The development of ICCs and electrical rhythmicity persists in vitro. ICCs appear to be necessary for the initiation of electrical rhythmicity. These findings provide further evidence for the pacemaker role of ICCs.