The dominant metabolic pathway of the presumably carcinogenic food antioxidant 2(3)-tert-butyl-4-hydroxyanisole (BHA) includes O-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) and subsequent peroxidation to 2-tert-butyl(1,4)paraquinone (TBQ). In order to determine the ability of TBHQ to induce the formation of oxygen radicals, electron spin resonance measurements were performed in presence and absence of peroxidases. ESR analyses showed that prostaglandin H synthase resulted in a substantially accelerated metabolism of TBHQ into TBQ, which is accompanied by formation of superoxide anion, hydroxyl radical and hydrogen peroxide. Spectrophotometric measurements revealed that prostaglandin H synthase and lipoxygenase are both capable of converting TBHQ into TBQ. In order to determine the effect of prostaglandin H synthase on BHA (dose-level: 1.5% BHA of the diet) metabolism in vivo, we coadministered two inhibitors of prostaglandin H synthase acetylsalicylic acid and indomethacin, with BHA to rats. Coadministration of acetylsalicylic acid (0.2%) in the drinking water resulted in a significant increase of urinary TBHQ excretion. Both acetylsalicylic acid and indomethacin (dose-level: 0.002% in the drinking water) induced a significant decrease in TBQ excretion into urine. Co-oxidation by prostaglandin H synthase of the BHA-metabolite TBHQ into TBQ, yielding reactive oxygen species might therefore be responsible for the carcinogenic and toxic responses elicited by this antioxidant.