BACKGROUND The effect of a medication on sphincter of Oddi motility should be characterized if it is to be used during sphincter of Oddi manometry. Controversy exists as to whether midazolam influences sphincter of Oddi motility. This study assessed the effect of midazolam on the hypertensive sphincter of Oddi. METHODS The study population consisted of 36 patients who presented with recurrent abdominal pain resulting from sphincter of Oddi dysfunction. The study was nonrandomized, prospective, and placebo controlled. Patient allocation was consecutive. Sphincter of Oddi manometry was performed in standard fashion. Manometric tracings were interpreted while the investigator was blinded to treatment allocation. Eighteen patients in the test group received 2 mg of midazolam intravenously whereas the 18 patients in the control group received saline solution intravenously. Manometric parameters were measured before and 3 minutes after the intravenous infusion. Changes in manometric findings before and after the administration of saline solution and midazolam were compared. RESULTS Midazolam caused a significant reduction in basal sphincter of Oddi pressure (24 mm Hg) as compared with saline solution (p < 0.001). Diagnostic concordance (normal vs. abnormal) between the basal sphincter pressure before and after midazolam was seen in only 77% of patients. CONCLUSIONS Midazolam significantly altered sphincter of Oddi motility. The decrease in sphincteric pressures would have altered diagnosis and management in 4 of 18 patients. Midazolam should not be used during sphincter of Oddi manometry.