In mammals that develop rectal aganglionosis, the aganglionic segment still exhibits spontaneous phasic contractions that contributes to dysmotility and pseudo-obstruction in this region. However, almost nothing is known about the mechanisms that generate these myogenic contractions, nor the effects of aganglionosis on the generation of Ca²⁺ waves that underlie contractions of the longitudinal muscle (LM) and circular muscle (CM). In a mouse model of Hirschsprung's disease (Ednrb^s-l/Ednrb^s-l ), the Ca²⁺ indicator fluo-4, was used to simultaneously monitor the temporal activation and spread of intercellular Ca²⁺ waves in LM and CM, during spontaneous colonic motor activities. During the intervals between colonic migrating motor complexes (CMMCs) in control mice, Ca²⁺ waves discharged asynchronously between the LM and CM. However, in these same mice, during CMMCs, a burst of discrete Ca²⁺ waves fired simultaneously in both muscle layers, where the propagation velocity of Ca²⁺ waves significantly increased, as did the rate of initiation and number of collisions between Ca²⁺ waves. Hexamethonium (300µM) or atropine (1µM) prevented synchronized firing of Ca²⁺ waves. In the aganglionic distal colon of Ednrb^s-l/Ednrb^s-l mice, not only were CMMCs absent, but Ca²⁺ waves between the two muscle layers fired asynchronously, despite increased propagation velocity. The generation of CMMCs in control mice involves synchronized firing of enteric motor nerves to both the LM and CM, explaining the synchronized firing of discrete Ca²⁺ waves between the two muscle layers. Aganglionosis results in a sporadic and sustained asynchrony in Ca²⁺ wave firing between the LM and CM and an absence of CMMCs.