We evaluated the intramural neural control of the opossum sphincter of Oddi (SO) in an in vitro preparation. Force transducers were used to record contractions at four sites along the sphincter segment. To stimulate intramural nerves, 10- to 120-s trains of pulses (4-10 V amplitude, 0.5 ms duration, and 5 Hz frequency) were delivered to one of three electrode pairs implanted along the SO. Electrical stimulation in the proximal, mid, or distal SO elicited phasic contractions that invariably originated in the proximal SO and propagated antegrade along the entire length of the sphincter segment. Stimulus-evoked contractions resembled spontaneous antegrade peristaltic contractions, but occurred at a higher rate (12-20/min). Atropine completely blocked this excitatory response to nerve stimulation. After atropine, nerve stimulation in the proximal, mid, or distal SO abolished spontaneous contractions at and distal to the site of stimulation for the duration of the stimulus. The inhibitory response to nerve stimulation was completely blocked by tetrodotoxin but was unaffected by phenoxybenzamine, tolazoline, or propranolol. We conclude that 1) the opossum SO is innervated by intramural cholinergic excitatory nerves and nonadrenergic noncholinergic inhibitory nerves; 2) cholinergic excitatory nerves are organized in ascending neural pathways, whereas nonadrenergic noncholinergic inhibitory nerves descend along the length of the SO; and 3) these neural pathways may modulate SO peristalsis in vivo and participate in ascending excitatory and descending inhibitory reflexes.