Double applications of phorbol eaters trigger excessive reactive oxygen species (ROS) production in mouse skin. previously reported data suggest that the two applications induce distinguishable biochemical events, namely, priming and activation. The former is characterized as a recruitment of inflammatory cells, such as neutrophils, by chemotactic factors to inflammatory regions and edema formation. The latter is responsible for ROS generation. Thus, inhibitory effects of 1'-acetoxychavicol acetate (ACA), previously reported to be a superoxide generation inhibitor is vitro, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammatory responses in mouse skin model were examined using a double application of ACA. We demonstrated that two pretreatments and pretreatment with ACA (810 mmol) in the activation phase suppressed double TPA application-induced H2O2 formation in mouse skin, ACA exhibited no inhibitory effects on edema formation and the enhancement of myeloperoxidase activity during the first TPA treatment, whereas the anti-inflammatory agent genistein administered at the same dose inhibited both biomarkers, No inhibitory potential of ACA for TPA-induced H2O2 formation in the priming phase was confirmed. On the other hand, in the in vitro study, ACA inhibited ROS generation in differentiated HL-60 cells more strongly than did 1'-hydroxychavicol, which showed no inhibition by pretreatment in the activation phase In addition, allopurinol did not inhibit double TPA application-induced H2O2 formation in mouse skin, These findings suggest that the NADPH oxidase system of neutrophils rather than the epithelial xanthine oxidase system is dominant for the O-2(-)-generating potential in double TPA-treated mouse skin. ACA significantly inhibited mouse epidermis thiobarbituric acid-reacting substance formation, known as an overall oxidative damage biomarker, Moreover, histological studies demonstrated that ACA inhibited double TPA treatment-induced morphological changes reflecting inflammatory response, such as edema formation, leukocyte infiltration, hyperplasia, and cell proliferation. Furthermore, pretreatment with ACA but not 1'-hydroxychavicol in the activation phase inhibits double TPA application-induced increases in both number of leukocytes and proliferating cell nuclear antigen index. These results suggested that ROS from leukocytes including O-2(-) plays an important role for continuous and excessive production of chemotactic factors, leading to chronic inflammation and hyperplasia, which are inhibitable by ACA. Thus, we concluded that O-2(-) generation inhibitors are agents that effectively inhibit oxidative stress and inflammatory responses in mouse skin.