We examined the pharmacological profile of ramosetron, a 5-HT₃-receptor antagonist for irritable bowel syndrome with diarrhea, comparing it with those of other 5-HT₃-receptor antagonists, alosetron and cilansetron, and the anti-diarrheal agent loperamide. Ramosetron showed high affinity for cloned human and rat 5-HT₃ receptors, with K_i values of 0.091 ± 0.014 and 0.22 ± 0.051 nmol/L, respectively, while its affinities for other receptors, transporters, ion channels, and enzymes were negligible. Dissociation of ramosetron from the human 5-HT₃ receptor was extremely slow (t_1/2 = 560 min), while alosetron (t_1/2 = 180 min) and cilansetron (t_1/2 = 88 min) dissociated relatively rapidly. Ramosetron competitively inhibited 5-HT-induced contraction of isolated guinea-pig colon, with pA₂ values of 8.6 (8.5 – 9.0). Ramosetron given orally also dose-dependently inhibited the von Bezold-Jarisch reflex in rats, with an ED₅₀ value of 1.2 (0.93 – 1.6) μg/kg. In addition, oral ramosetron dose-dependently inhibited restraint stress-induced defecation in rats, with an ED₅₀ value of 0.62 (0.17 – 1.2) μg/kg. In all of these experiments, the potencies of ramosetron were greater than those of alosetron, cilansetron, or loperamide. These results indicate that ramosetron is a highly potent and selective 5-HT₃-receptor antagonist, with beneficial effects against stress-induced abnormal defecation in rats.