This study investigated the involvement of tryptase and proteinase-activated receptor (PAR) subtypes in spontaneous scratching, an itch-associated behavior, in NC mice. This strain of mice showed chronic atopy-like dermatitis and severe spontaneous scratching, when kept a long time in a conventional environment. The trypsin-like serine proteinase inhibitor nafamostat mesilate (1 – 10 mg/kg) dose-dependently inhibited spontaneous scratching in mice with dermatitis. The activity of tryptase was increased in the lesional skin, which was inhibited by nafamostat at a dose inhibiting spontaneous scratching. Enzyme histochemistry revealed the marked increase of toluidine blue–stained cells, probably mast cells, with tryptase activity in the dermis of the lesional skin. Intravenous injection of anti-PAR₂ antibody suppressed spontaneous scratching of mice with dermatitis. Intradermal injection of the PAR₂-activating peptide SLIGRL-NH₂, but not PAR₁, ₃, ₄-activating peptides, elicited scratching at doses of 10 – 100 nmol/site in healthy mice. PAR₂-immunoreactivity was observed in the epidermal keratinocytes in healthy and dermatitis mice. These results suggest that PAR₂ and serine proteinase(s), mainly tryptase, are involved in the itch of chronic dermatitis.