BACKGROUND: Both ondansetron and granisetron exert their antiemetic effects via a blockade of the 5-hydroxytryptamine 3 receptor (5-HT₃R). Because the 5-HT₃R is a member of a superfamily of ligand-gated ion channels and has structural similarities to the nicotinic acetylcholine receptor (nAChR), a 5-HT₃R antagonist may also inhibit the nAChR. This study examined the effects of 5-HT₃R antagonists, ondansetron and granisetron, on rocuronium-induced neuromuscular blockade in vitro. METHODS: Rat phrenic nerve-hemidiaphragm preparations were isolated and allocated randomly into seven groups (control, 1, 10, 100 µgram/ml of ondansetron, 0.1, 1, 10 µgram/ml of granisetron). Two studies were carried out using single twitch responses. In the cumulative dose-response study, rocuronium 1 µgram/ml and each doses of ondansetron or granisetron were administered simultaneously, and incremental 0.5 µgram/ml doses of rocuronium were added to obtain more than 95% neuromuscular twitch inhibition. ED₅, ED₅₀, ED₉₀, and ED₉₅ of rocuronium in each group were calculated using a logistic model. In the partial curarization study, the twitch heights were measured after administering ondansetron or granisetron (10 minutes after administering 3 µgram/ml rocuronium) and were measured 10 minutes later. The 2 twitch heights were then compared. RESULTS: In the cumulative dose-response study, ondansetron 100 µgram/ml and granisetron 10 µgram/ml significantly reduced the ED₅₀ of rocuronium (P < 0.05). There were no intergroup differences in the partial curarization study. CONCLUSIONS: High concentration of ondansetron and granisetron enhanced the neuromuscular blockade of rocuronium. Granisetron enhanced the neuromuscular blockade of rocuronium more potently than ondansetron.