The characteristics of the ileo-junction (UIC) were examined in seven opossums in vivo, and the effect of the UIC on colo-ileal reflux in eight opossums in vitro. Electromyography and intraluminal manometry were studied during intestinal distensions, and administration of phenylephrine, isoproterenol and carbachol. In vitro studies used preparations of ileum, UIC, and colon, attached to a propulsion evaluation system. Fluid flow across the UIC was studied basally and after phenylephrine, isoproterenol and carbachol. A high pressure zone in the UIC was not observed in vivo. Colonic distension increased the pressure and electrical spike bursts in the ileum and UIC, while ileal distension had the opposite effect. Myoelectric and contractile activities were inhibited by adrenergic agonists and stimulated by carbachol. In vitro studies demonstrated aborally migrating ileal contractions initiated by fluid injections into the ileum, and cecal contractions elicited by fluid injections into the colon. The UIC only prevented colo-ileal reflux when it was undergoing contraction as part of ileal or colonic activity. These findings suggest that the opossum UIC does not have valvular properties and ileal fluid propulsion is the main factor in the prevention of colo-ileal reflux.