Midazolam is a more recent benzodiazepine used for sedation during endoscopic procedures, including sphincter of Oddi (SO) manometry. However, the effect of this drug on the human SO has so far not been studied. In this paper we explored the effect of midazolam on human SO motility by means of endoscopic manometry. Twelve patients with suspected SO dysfunction were investigated. We found that in patients with normal manometry findings, midazolam had no effect on the sphincter motility. In contrast, in all patients with elevated SO pressure (SOP), as well as in one of the three patients with borderline SO tone, midazolam (2.5 mg i.v.) produced a relaxatory effect. Due to this effect the final readings were affected in three out of five patients, i.e. the abnormally elevated SOP decreased to a borderline level in two and in the remaining patient the borderline SOP decreased to normal. In one other patient with a markedly elevated SOP the additional injection of 2.5 mg of midazolam caused a further decrease in SOP to a borderline level. We conclude that midazolam, due to its relaxatory effect on SO, appears to be a less suitable sedative for SO manometry.