Intrinsic neurons containing serotonin (5-HT) are involved in the regulation of gastrointestinal motor function and are also thought to be important in the modulation of visceral sensory function. We have evaluated the effect of a specific 5-HT3 antagonist (ondansetron, O) on visceral sensation and rectal compliance in a randomized, double-blind, cross-over, placebo (P) controlled study of O 16 mg 3 times/day, in healthy volunteers and patients with irritable bowel syndrome (IBS). Symptoms were also evaluated in the latter group. A 2-week run-in period was followed by two 2-week treatment arms of P and O, separated by a 2-week wash-out period. Twelve healthy subjects and 9 patients with IBS were recruited. Assessment was by daily symptom and bowel function diary, and physiological tests of anal manometry, rectal sensory testing to distension and electrical stimulation, and rectal compliance. Ten healthy subjects completed the entire study, and 6 IBS patients completed the diary card evaluation, including 5 who also completed the physiological evaluation. O caused significantly (p < 0.01) firmer stools when considering both subject groups together. In the healthy subjects no physiological parameters were altered by O. In IBS patients the rectal sensory threshold to electrical stimulation tended to increase with O (20 vs. 28 mA, P vs. O, median, p = 0.06) while the urge (80 vs. 60 ml, p = 0.05) and maximum tolerated volumes (130 vs. 90, p = 0.03) to distension tended to decrease with O. Patients with IBS experienced significantly fewer daily episodes of pain while on O (2 vs. 1, p = 0.03). Serotonin-3 antagonism (O) causes firmer bowel actions in all subjects, and may affect gut sensitivity and pain in patients with IBS.