The prostaglandin H synthase (PHS)-catalyzed metabolism of indenestrol A (IA), indenestrol B (IB) and indanestrol (I) and the effects of these compounds on PHS were studied in incubations with ram seminal vesicle microsomes (RSVM) by means of arachidonic acid (20:4)-dependent oxygen consumption and by HPLC analysis of parent compound conversion as well as UV spectroscopy. IA and I were metabolized by PHS via co-oxidation. By analogy with diethylstilbestrol (DES) they stimulated PHS cyclo-oxygenase dose-dependently and became inhibitory at higher concentrations. Cyclo-oxygenase activity determined at 20:4 concentrations ranging from 10 to 70 microM revealed an antioxidant-type of inhibition for IA with IC50 values ranging from 30 to 150 microM. IB, on the other hand, displayed an indomethacin-like type of PHS inhibition with an IC50 value of 20 microM not dependent upon 20:4 concentration which was consistent with the observation that IB inhibited the co-oxidation of DES when initiated with 20:4 but not with hydrogen peroxide. Recovery of IB was incomplete in extracts from incubations with native PHS, but the reaction was neither 20:4 dependent nor inhibited by indomethacin or catalase; it was partially inhibited by eicosatetraynoic acid (ETYA) or by butylhydroxyanisol (BHA). This may indicate affinity of IB for the enzyme protein and conversion of IB other than by a co-oxidation mechanism. UV spectroscopy revealed the formation of a p-quinoid intermediate in incubations with IA, but not with IB or I. The IA-quinone was synthesized and reacted with nucleophiles such as water, methanol, ethanol and mercaptoethanol to adducts which were further characterized by gas chromatography/mass spectrometry. Our data indicate that indanyl derivatives of DES interact differently with PHS and thus could provide a useful tool for future studies on the mechanism of action of tumorigenic stilbene estrogens as well as on the elucidation of the role of PHS-mediated metabolism in their toxic action.