Background: Serine protease promotes desquamatation of the stratum corneum and this is controlled by serine protease inhibitors (SPI). After disruption of the skin barrier, signals for barrier recovery are started with the activation of cytokines and a migration of calcium ions. On the other hand, the protease-activated receptor-2 (PAR-2) pathway is initiated as a negative signal. As the pH of the stratum corneum become neutral, activated serine protease and PAR-2 inhibit the secretion of lamellar bodies and the formation of the lamellar structure. Objective: We wanted to screen noble synthetic peptides and identify the efficacy of a selected peptide, Palmitic acid-Lysine Threonine Threonine Lysine Serine (Pal-KTTKS), on PAR-2 in vitro and in vivo, and a clinical study was performed. Methods: In vitro: Changes of the intracellular calcium ion concentration were measured in cultured HaCaT cells by fluorescence imaging according to treatment with sample peptides and trypsin. In vivo animal study: The efficacy of 2% Pal-KTTKS cream as a selected noble peptide was evaluated in an oxazolone-induced atopic dermatitis animal model. Clinical study: A total of twenty three atopic dermatitis patients applied 2.5% Pal-KTTKS peptide-containing cream on the one side of their extremities and pseudo-ceramide containing moisturizer on the other side of the extremities as a control twice a day for 4 weeks. Clinical improvement was evaluated by the Eczema Area Severity Index (EASI) score, a subject questionnaire and comparison of photographs. Results: Suppression of the intracellular calcium concentration via PAR-2 inhibition was noted in the Pal-KTTKS peptide treated cultured HaCaT cells. In the oxazolone-induced atopic dermatitis hairless mice model, 2% Pal-KTTKS peptide containing lotion was more effective than vehicle lotion only. In the atopic dermatitis patients, the sites treated with 2.5% Pal-KTTKS peptide-containing cream showed better improvement for the EASI score, the subject questionnaire and the clinical photographs as compared to that of the control sites. There were no remarkable side effects related to the treatment. Conclusion: A PAR-2 inhibitor-containing topical agent would be an effective and safe modality for treating atopic dermatitis. (Korean J Dermatol 2010;48(11):966∼974)