In gastrointestinal smooth muscles, both M₂ and M₃ subtypes of muscarinic receptors are co-expressed with a preponderance of the former subtype. Many previous studies have suggested that M₃ receptors exclusively contribute to the gastrointestinal smooth muscle contractions. However, the precise roles of the M₂ receptors in the regulation of gut motility remain to be elucidated. In the present study, we simultaneously recorded changes in the intraluminal pressure (IP), longitudinal tension (LT), and propelled volume (PV) in isolated colonic segments from M₂ receptor knockout (KO) and wild-type (WT) mice. In the WT preparations, luminal distension induced a continuous rhythmic contractile activity that was characterized by synchronous rises in IP and LT, occurring periodically at a constant interval. When atropine was applied, the frequency of rhythmic contractile activity was unchanged, but the amplitude of IP and PV were significantly reduced. Application of tetrodotoxin or hexamethonium abolished the rhythmic contractile activity. In the M₂KO preparations, the frequency of contractile activity elicited by luminal distension was comparable to that in WT preparations. However, the amplitude of IP and PV were significantly reduced in M₂KO preparations. These results suggest that M₂ muscarinic receptors participate in the regulation of colonic motor activity and have a significant role in propel of luminal contents.