This study involved immunohistochemistry and intracellular electrophysiology to investigate serotonergic neurotransmission in the sphincter of Oddi (SO). 5-Hydroxytryptamine (HT)-positive neurons (14 cells/preparation) and nerve fibers were observed in the ganglionated plexus. Serotonergic nerve fibers, which persisted under 2- to 6-day organ culture, were densely distributed, with varicose endings encircling some SO neurons. When 5-HT was applied to SO neurons, it elicited three different responses: 1) a fast depolarization to 5-HT in 31 of 62 cells was mimicked by 2-methyl-5-HT and blocked by LY-278584 (1 microM); 2) a prolonged depolarization to 5-HT in 21 of 62 cells evoked an increase in input resistance and was attenuated by the 5-HT1P antagonist renzapride (1 microM) but not by the 5-HT4 antagonist SDZ-205557 (0.1-10 microM); and 3) an indirect depolarization blocked by TTX or atropine was observed in 32 of 62 cells. 5-HT superfusion elicited a dose-dependent monophasic depolarization (EC50 = 2 microM, n=14). In conclusion, 5-HT is present in nerves of the SO and elicits both 5-HT3 and 5-HT1P receptor-mediated depolarizations, supporting the concept that 5-HT plays a role in SO regulation.