Compound 48/80 induced scratching behavior in BALB/c mice, and the role of mast cell mediators in this behavior was examined. Mouse scratching behavior was detected and evaluated using a new apparatus, MicroAct. Compound 48/80 increased the incidence of scratching behavior and scratching time in a dose-dependent manner, accompanied by a potent activation of mast cells and a potent increase in vascular permeability. Dibucaine and μ-opioid receptor antagonists inhibited the scratching behavior. Although histamine H₁ receptor antagonists potently inhibited the vascular permeability increase, they did not affect the scratching behavior. Methysergide inhibited the scratching behavior slightly without affecting the vascular permeability increase, whereas cyproheptadine inhibited both. A cyclooxygenase inhibitor, a 5-lipoxygenase-activating protein inhibitor and a PAF receptor antagonist did not affect the scratching behavior. High doses of serotonin induced scratching behavior less frequently than did compound 48/80. Furthermore, mast cell-deficient WBB6F1-W/W^v mice exhibited frequent scratching behavior after injection of compound 48/80. These results clearly indicate that compound 48/80 can induce scratching behavior in mice independent of mast cell mediators.