DiI-retrograde labeling and intracellular electrophysiological techniques were used to investigate the mechanisms underlying the generation of spontaneously occurring colonic migrating myoelectric complexes (colonic MMCs) in mice. In isolated, intact, whole colonic preparations, simultaneous intracellular electrical recordings were made from pairs of circular muscle (CM) cells during colonic MMC activity. During the intervals between colonic MMCs, spontaneous inhibitory junction potentials (IJPs) were always present. The amplitudes of spontaneous IJPs were highly variable (range 1-20mV) and occurred asynchronously in the two CM cells, when separated by 1mm in the longitudinal axis. Colonic MMCs occurred every 151 ± 7sec in the CM and consisted of a repetitive discharge of cholinergic rapid oscillations in membrane potential (range: 1-20mV) that were superimposed on a slow membrane depolarization (mean amplitude: 9.6 ± 0.5mV; half duration: 25.9 ± 0.7sec). During the rising (depolarizing) phase of each colonic MMC, cholinergic rapid oscillations occurred simultaneously in both CM cells, even when the two electrodes were separated by up to 15mm along the longitudinal axis of the colon. Smaller amplitude oscillations (<5mV) showed poor temporal correlation between two CM cells, even at short electrode separation distances (i.e. <1mm in the longitudinal axis). When the two electrodes were separated by 20mm, all cholinergic rapid oscillations and IJPs in the CM (regardless of amplitude) were rarely, if ever, coordinated in time during the colonic MMC. Cholinergic rapid oscillations were blocked by atropine (1µM) or tetrodotoxin (1µM). Slow waves were never recorded from any CM cells. DiI labeling showed that the maximum projection length of CM motor neurons and interneurons along the bowel was 2.8mm and 13 mm, respectively. When recordings were made adjacent to either oral or anal cut ends of the colon, the inhibitory or excitatory phases of the colonic MMC were absent, respectively. In summary, during the colonic MMC, cholinergic rapid oscillations of similar amplitudes occur simultaneously in two CM cells separated by large distances (up to 15mm). As this distance was found to be far greater than the projection length of any single CM motor neuron, we suggest that the generation of each discrete cholinergic rapid oscillation represents a discreet cholinergic excitatory junction potential (EJP), that involves the synaptic activation of many cholinergic motor neurons simultaneously, by synchronous firing in many myenteric interneurons. Our data also suggests that ascending excitatory and descending inhibitory nerve pathways interact and reinforce each other.