We investigated the role of serotonin (5-hydroxytryptamine; 5-HT) 2 and 5-HT 3 receptor subtypes in acute itch-associated scratching behavior as well as in an allergic pruritus model in rats. Intradermal 5-HT evoked hind limb scratching directed toward the injection site in naïve rats. Scratching behavior was significantly reduced by pretreatment with the 5-HT 2 receptor antagonist ketanserin. Intradermal injection of ␣-methylserotonin, a 5-HT 2 receptor agonist, also elicited scratching behavior in a dose-dependent manner, indicating that acute 5-HT-induced scratching is mediated via peripheral 5-HT 2 receptors. To produce a model of allergic pruritus, skin was sensitized by topical application of 5% dinitrofluorobenzene (DNFB). One month later, repeated challenge of the skin with 0.2% DNFB at weekly intervals elicited scratching as part of the immediate allergic response. Scratching was not affected by ketanserin or by the 5-HT 3 receptor antagonist ondansetron, indicating that neither 5-HT 2 nor 5-HT 3 receptors is involved in itch-associated scratching behavior caused by allergic skin dermatitis in rats.