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Sphincter of Oddi (SO) dysfunction as a potential cause of chronic acalculous cholecystitis (CAC) has not been studied in cases for which intraoperative SO manometry was used during laparoscopic cholecystectomy. In this study, we evaluated the effects of carbon dioxide pneumoperitoneum on laparoscopic transcystic SO manometry. In 27 patients with CAC, transcystic SO manometry had been attempted during laparoscopic cholecystectomy. The mean age of the patients was 46 years (range, 22–71). Complete manometric data sets were obtained in 18 patients. The mean SO pressure, phasic SO pressure, and phasic frequency were 35.4 ± 29.1 mm/Hg versus 30.8 ± 23.8mm/Hg, 104.8 ± 63.0 mm/Hg versus 73.6 ± 34.6 mm/Hg, and 2.1 ± 1.8 contractions/min versus 2.8 ± 3.4 contractions/min with and without pneumoperitoneum, respectively. All differences were nonsignificant (P > 0.05). Two complications (7.4%) were observed: pancreatitis and jaundice. SO manometry is not affected by CO2 pneumoperitoneum. It may be used to study SO motility in patients with CAC.