The metabolism in mouse epidermis of benzo[b]fluoranthene (BbF) was studied. [3H]BbF was applied topically, mice were killed at various intervals, and metabolites were extracted from the epidermis and analyzed by h.p.l.c The major metabolites were identified by comparisons to standards as 4-, 5-, and 6-hydroxyBbF. Sulfate and glucuronide conjugates of these hydroxyBbF were also detected. Minor metabolites included 12-hydroxyBbF, BbF-1,2-diol, and BbF-11,12-diol. BbF-9,10-diol, the only known tumorigenic oxygenated derivative of BbF, was not detected. The further metabolism of BbF-9,10-diol was studied in vitro, using rat liver 9000 g supernatant. The major metabolites were identified by their spectral characteristics as 5-and 6-hydroxyBbF-9,10-diol. Little if any BbF-9,10,11,12-tetraol was detected. 5-and 6-HydroxyBbF-9,10-diol were not detected as metabolites of [3H]BbF in mouse epidermis. Several known and potential BbF metabolites-BbF-1,2-diol, BbF-11,12-diol, BbF-9,10-diol, BbF-9,10-diol-11, 12-epoxide, 5-and 6-hydroxyBbF-9,10-diol, 1-hydroxyBbF, 5-hydroxyBbF, and 6-hydroxyBbF-were tested for tumor initiating activity on mouse skin. Among these, only BbF-9,10-diol showed high tumorigenic activity, but no evidence has been obtained for its formation in vivo from BbF. These studies do not support the hypothesis that BbF is metabolically activated through formation of the bay region diol epoxide, BbF-9,10-diol-11,12-epoxide.