The microsomal fraction of bovine vesicular gland catalyzed the conversion of [1-(14)C]8,11,14-eicosatrienoic acid to prostaglandin E(1) in the presence of tryptophan, hemoglobin, and glutathione. The prostaglandin synthetase system was solubilized by treatment of the microsomal fraction with Tween 20 in the presence of ethylene glycol. DEAE-cellulose column chromatography separated the enzyme into two fractions (Fractions I and II), both of which were required for prostaglandin E(1) synthesis. When Fraction I alone was incubated with 8,11,14-eicosatrienoic acid, an unstable compound accumulated. This compound was converted to prostaglandin E(1) by the addition of Fraction II. On the basis of its R(F) values on thin-layer chromatography, its reduction to prostaglandin F(1alpha) with stannous chloride, and the decomposition to prostaglandins F(1alpha), E(1), and D(1) at room temperature, the unstable intermediate was tentatively identified to be the 9,11-endoperoxide derivative, referred to previously as prostaglandin R(1) (Nugteren et al. (1973) Biochim. Biophys. Acta 326, 448-461) or prostaglandin H(1) (Hamberg et al. (1974) Proc. Nat. Acad. Sci. USA 71, 345-349). Of the three cofactors mentioned above, Fraction I required both tryptophan and hemoglobin, while only glutathione was necessary for Fraction II. Anti-inflammatory agents such as indomethacin and aspirin were inhibitory to Fraction I.