Skin sulfhydryl oxidase (SSO) was prepared from cow snout skin. Dithiothreitol was the thiol substrate most efficiently utilized, with 2-mercaptoethanol, glutathione, and cysteine having less than 23% the relative efficiency of dithiothreitol. In the presence of SSO reductively denatured ribonuclease A was reoxidized and reactivated. The degree of reactivation was proportional to the incubation time and dependent on the amount of enzyme added. The oxidation of thiols by SSO required molecular oxygen as an oxidant. The oxygen consumption increased linearly with the increasing concentration of SSO during the increasing formation of disulfide from sulfhydryl groups. The activity of SSO was detected in differing amounts in each of the four layers--stratum corneum, stratum granulosum, stratum spinosum with basal cell layer, and dermis--of cow snout skin, while showing the highest specific activity in the stratum granulosum.