1-Phenylazo-2-hydroxynaphthalene (Sudan I) is converted by microsomal enzymes of rat livers in vitro to 5 products. Hepatic microsomes from 5,6-benzoflavone-treated rats are more effective for the metabolism of Sudan I than those from phenobarbital- or Sudan I alone-treated rats. Major products formed by microsomes are identified as the ring-hydroxyderivatives of benzene and naphthalene rings. The formation of the benzenediazonium ion evolved by oxidative splitting of the azo group of Sudan I by microsomal enzymes is also proved. The oxidative splitting of Sudan I by microsomal enzymes may be considered as the possible mechanism of the Sudan I activation to the ultimate carcinogen (benzenediazonium ion).