The circular smooth muscle of the rabbit internal anal sphincter, tested in vitro, exhibited spontaneous resting tonus and relaxed with neural stimuli of 0.05-0.5-ms duration and 1-10-Hz frequency. The relaxation was abolished by tetrodotoxin (10(-6) M) but not by atropine or propranolol at the same molar concentration, suggesting that the inhibitory neural fibers mediating relaxation are nonadrenergic and noncholinergic. One-hour incubation in physiologic solution containing 8% of rabbit vasoactive intestinal peptide (VIP) antiserum significantly reduced the relaxation induced by electrical stimulation. The reduction was greater for the shorter duration stimuli, ranging from 80% to 50% for 0.05-ms stimuli to 35%-11% for 0.5-ms stimuli. Control strips were relaxed in a dose-dependent fashion by 10(-9)-10(-6) M VIP; the antiserum at the concentration used completely blocked the relaxation produced by 10(-7) M VIP and reduced the relaxation produced by 10(-6) M VIP. Adenosine triphosphate also relaxed the internal anal sphincter in a dose-dependent manner. Prolonged inhibitory nerve stimulation (0.5 ms, 10 Hz, 30-min train) caused significant reduction in the relaxation induced by exogenous VIP, whereas the relaxation induced by adenosine triphosphate was unaffected. These data are consistent with VIP being an inhibitory neurotransmitter responsible for relaxation of the internal anal sphincter.