The mechanism involved in the reaction catalyzed by skin sulfhydryl oxidase was investigated. Skin sulfhydryl oxidase, which was purified to a 300-fold increase in specific activity over rat skin, was used. As the substrate for the reaction catalyzed by skin sulfhydryl oxidase, dithiothreitol (DTT) was used. The disappearance of DTT, consumption of O2 and production of H2O2 were measured according to Takamori's Janolino's and Kakinuma's methods, respectively. The molar ratio of the disappearance of DTT and the consumption of O2 during the enzyme reaction was 1:1.05. The molar ratio of the disappearance of DTT and the H2O2 production during the enzyme reaction was 1:086. Results therefore showed that the molar ratio of the disappearance of DTT, consumption of O2, and production of H2O2 was 1:1:1. Therefore, the stoichiometry of the enzyme reaction was estimated to be (formula; see text)