The Ussing chamber is often used for in vitro investigations into the transport physiology of epithelia including the small intestine. However, the morphological and functional integrity of the small intestine incubated in an Ussing chamber has been largely ignored. The present study attempted to compare the mucosal injury among different segments of the mouse small intestine when incubated in an Ussing chamber. To assess the functional damage, we measured changes in the transmural potential difference (PD) evoked by adding glucose to the mucosal solution and forskolin to the mucosal and serosal solutions. Morphological deterioration was assessed histologically. The villi in the duodenum and proximal jejunum were morphologically damaged by 2 h of incubation in an Ussing chamber and almost completely destroyed within 4 h, while crypts remained intact. The villi were moderately damaged in the distal jejunum. In contrast, the integrity of the villi and crypts was maintained in the ileum for 4 h. The basal PD and forskolin-induced PD were maintained up to 4 h of incubation in all segments. On the other hand, the glucose-induced PD was not apparent in the duodenum at 0 h, and was gradually suppressed to zero in the proximal jejunum by 4 h, although the glucose-induced PD was maintained for 4 h in the ileum. The loss of villous epithelial integrity was correlated with the disappearance of the glucose-induced PD, while both the basal and forskolin-induced PD were retained, even in the tissue with disrupted villi. It is concluded that the mucosa of the proximal small intestine was rapidly injured in the Ussing chamber, particularly in the villi, while the integrity of the mucosa of the distal small intestine was maintained for 4 h. The glucose-induced PD, but not the basal or forskolin-induced PD, can be used as a marker of the villous integrity.