During microsomal metabolism of [14C]naphthalene, radioactive material was irreversibly bound to microsomal protein. The binding seems to result predominantly from secondary oxidation of naphthol rather than from primary monooxygenation of the hydrocarbon. This is supported by the following observations: a) The metabolism of naphthalene was rapid, e.g., 100 nmoles were metabolized within 10 min, whereas protein-bound radioactivity increased for at least 80 min. b) Inhibition of the epoxide hydrase by trichloropropene oxide did not significantly affect the binding. c) Addition of UDP-glucuronic acid to the incubation mixture reduced the binding, presumably by conjugation of the phenolic metabolites. d) During microsomal metabolism of [14C]1-naphthol, a major metabolite of [14C]naphthalene, a considerable amount of protein-bound radioactivity was found, indicating the formation of reactive metabolites. e) The addition of unlabeled 1-naphthol decreased the binding of [14C]naphthalene; this may be attributed to a dilution of the [14C]labeled naphthol-pool derived from [14C]naphthalene. Thus, the results are in agreement with our previous observations on the binding studies of 2,2'-dichlorobiphenyl and suggest that the major part of protein-bound metabolites of some aromatic hydrocarbons, formed in vitro, derive from secondary metabolism of the phenol(s).