5,5-Diphenylhydantoin irreversibly binds to rat liver microsomes and the process requires NADPH and O2. Protein binding was significantly enhanced when experiments were carried out with liver microsomal preparations from β-naphthoflavone and 3-methylcholanthrene pretreated animals whereas pretreatment with phenobarbital significantly reduced it. Carbon monoxide, β-diethylaminoethyl-diphenylpropylacetate and glutathione inhibited drug covalent binding to microsomal proteins. In contrast, enhanced drug binding was observed when trichloropropene oxide and cyclohexene oxide, two epoxide hydrolase inhibitors, were added to the incubation mixture. 5,5-Diphenylhydantoin in vitro metabolism was quantitatively determined by gas liquid chromatography with selected ion monitoring. A good correlation seems to exist between drug covalent binding and the microsomal process of 5,5-diphenylhydantoin hydroxylation to 5-(4-hydroxyphenyl)-5-phenylhydantoin. The results presented support a previous hypothesis on the intermediacy of arene oxides in the biotransformation of this drug. © 1982.