A number of studies have demonstrated that 5-hydroxytryptamine (5-HT) can induce muscle contraction or relaxation response and enhance secretion in the gastrointestinal tract via a multiplicity of 5-HT receptor subtypes. In the present study, we investigated the pharmacological characterization of the 5-HT-induced contractile response in longitudinal smooth muscle isolated from the feline ileum. Addition of 5-HT into muscle chambers enhanced the basal tone and spontaneous activity in a concentration-dependent manner. The neurotoxin tetrodotoxin did not alter the 5-HT-induced contraction of the longitudinal muscles. Neither atropine nor guanethidine affected the contraction. The 5-HT agonists, 5-methylserotonin hydrochloride and mosapride, also evoked concentration-dependent contractions. The 5-HT-induced contraction was enhanced by the 5HT2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist ondansetron but was inhibited by the 5-HT1 receptor antagonist methysergide and 5-HT4 receptor antagonist GR113808. These results indicate that 5-HT1 and 5-HT4 receptors may mediate the contraction of the 5-HT-induced response and 5-HT2 and 5-HT3 receptors may mediate 5-HT-induced relaxation in feline ileal longitudinal smooth muscles.