Our experiments were conducted to determine whether substance P (SP) would elicit an itch sensation mediated by mast cells in mice. An intradermal injection of SP (10–135 μg site⁻¹) into the rostral back of the ICR mouse dose-dependently produced scratching of the injected site. The SP- (135 μg site⁻¹ = 100 nmol site⁻¹) induced scratching was inhibited by capsaicin (repeated administration) and naloxone; features being similar to itch in humans. SP elicited scratching in mast cell-deficient (WBB6F1W/W ^v) mice as well as control (+/+) mice. Pretreatment with compound 48/80 produced similar degrees of inhibition of SP-induced scratching in mast cell-deficient mice as well as control +/+ and ICR mice. Intradermal injections of the NK₁receptor agonist GR73632 produced dose-dependent scratching, while the NK₂ agonist GR64349 and the NK₃ agonist senktide were without effects. SP-induced scratching was inhibited by the NK₁ receptor antagonists spantide and L-668,169, but not by the NK₂ antagonist L-659,877. The results suggest that scratching of the mouse induced by an i.d. injection of SP is itch-associated response. The SP action may be mediated at least partly by cutaneous NK₁ receptors, and mast cells may not be key factors in SP-induced itching.