AbstractProstaglandin H synthase (PHS) has gained interest as a drugmetabolizing enzyme and has been shown to cooxidize and metabolically activate diethylstilbestrol (DES) in vitro. Both 7,8‐benzoflavone (α‐naphthoflavone, ANF) and 5,6‐benzoflavone (β‐naphthoflavone, BNF) have now been studied for their effects on PHS from ram seminal vesicle microsomes by means of several in vitro assays.The PHS‐catalyzed cooxidation of DES, as measured by high‐performance liquid chromatography (HPLC) analysis, is inhibited by BNF and ANF at micromolar concentrations, with median inhibitory concentrations (IC‐50) of<20 and 40 μM, respectively. The oxidation of DES is inhibited whether it is initiated by arachidonic acid or by hydrogen peroxide, indicating that the benzoflavones inhibit PHS by a mechanism different from that of indomethacin. Monitoring of cyclooxygenase activity in an oxygraph also reveals an inhibition of PHS by BNF which depends only weakly on arachidonic acid concentration; inhibition by ANF is less pronounced under these conditions. Since PHS‐catalyzed conversion of the benzoflavone compounds was detected under conditions permitting cooxidation, the inhibition of PHS by benzoflavones in vitro could either be a direct effect or possibly mediated via metabolites.Our data imply that ANF and BNF, in addition to their well‐known role as modifiers of mixed‐function oxidases, can affect the PHS‐catalyzed metabolism of xenobiotics. This is discussed in the context of adverse effects caused by DES in vivo and in cell culture and must be taken into account when interpreting the modifying effect of benzofla