7,8-Benzoflavone(ANF) is a potent in vitro inhibitor of CYP1A2 but is an in vitro activator of CYP3A4. We have investigated the inhibition of caffeine 3-demethylation by metabolites of ANF as well as ANF by human liver microsomes. ANF was the most potent among all the compounds tested. Metabolites of ANF with dihydrodiol substitution at positions 5,6 or 7,8 showed less inhibitory activity. These results suggest that ANF lies in the most appropriate orientation to the active site of CYP1A2. The activation of CYP3A4 enzyme activities by ANF and its metabolites was also investigated. Testosterone 6 beta-hydroxylation mediated by CYP3A4 was stimulated by ANF and metabolites with substitutions at positions 5,6 or 7,8. Hydroxy ANF metabolites, however, decreased the testosterone 6 beta-hydroxylation.