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Introduction: The serotonin 3A receptor (5-HT3AR) is involved in vomiting and gastrointestinal motility. However, it is not well understood the expression pattern of 5-HT3AR in the gut immunohistochemically and how much contribution of 5-HT3AR to upper or lower intestinal motility. Objectives: We investigated the contribution of 5-HT3AR to gastrointestinal motor function by using 5-HT3AR KO mice and sought to identify 5-HT3AR-expressing cells via immunohistochemical staining using 5-HT3AR-GFP reporter mice. Methods: The expression of 5-HT3AR was measured in each section of the gut through real-time PCR. The motor function of the stomach and colon was assessed via the 13C-octanoic acid breath test and colonic bead expulsion test, respectively, using 5-HT3AR KO mice. 5-HT3AR-expressing cells in the muscle layer of the gut were identified by immunohistochemical staining using 5-HT3AR-GFP reporter mice. Results: 5-HT3AR was expressed throughout the digestive tract, and 5-HT3AR expression in the stomach and lower digestive tract was higher than that in the other sections. Motor function in the stomach and colon was lower in 5-HT3AR KO mice than in WT mice. As a result of immunohistochemical staining using GFP reporter mice, cholinergic neurons and PDGFRα+ cells were shown to express 5-HT3AR. In contrast, 5-HT3AR indicated by GFP fluorescence was rarely detected in ICC and smooth muscle cells. Conclusions: These results show that 5-HT3AR is highly expressed in the stomach and large intestine and that the activation of 5-HT3AR accelerates gastric emptying and large intestine transit. Additionally, 5-HT3AR is highly expressed in cholinergic neurons and some interstitial cells, such as PDGFRα+ cells. [ABSTRACT FROM AUTHOR]