3-tert-Butylhydroxyanisole (3-BHA) is an antioxidant which can have a modulating effect on chemical carcinogenesis. Information concerning the metabolism of 3-BHA is incomplete. In the present study, the metabolites formed by incubating 3-BHA with liver microsomes from rats given beta-naphthoflavone by p.o. intubation were studied. Three metabolites were identified, two major metabolites and a minor metabolite. One of the major metabolites was the catechol of 3-BHA, i.e., 3-tert-butyl-4,5-dihydroxyanisole, which has not previously been reported. A characteristic of this compound is its capacity to be oxidized readily. The second major metabolite was tert-butyl hydroquinone which has been reported previously to be a liver microsomal metabolite of 3-BHA. The third metabolite, which occurred in small quantities, was 2,2'-dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl. 2,2'-Dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl has been identified previously as a major metabolite of 3-BHA in the rat intestine. An understanding of the metabolism of 3-BHA may assist in elucidating the mechanism(s) of its biological effects.