The prostaglandin synthetase system of bovine vesicular gland microsomes was solubilized and separated into Fractions I and II. The former fraction catalyzed the conversion of 8,11,14-eicosatrienoic acid to prostaglandin H1 (9 alpha, 11alpha-epidioxy-15(S)-hydroxy-13-trans-prostenoic acid). This compound was isomerized to prostaglandin E1 (11alpha, 15(S)-dihydroxy-9-keto-13-trans-prostenoic acid) by the action of Fraction II (Miyamoto, T., Yamamoto, S., and Hayaishi, O. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3645-3648). Fraction I was further purified by isoelectric focusing and about a 700-fold purification was achieved starting from the microsomes. When the enzyme was incubated with 8,11,14-eicosatrienoic acid in the presence of hematin, an unstable compound which was distinguishable from prostaglandin H1 accumulated. The chemical properties of this compound were identical with those of prostaglandin G1 (9 alpha, 11 alpha-epidioxy-15(S)-hydroperoxy-13-trans-prostenoic acid). The enzyme also catalyzed the conversion of prostaglandin G1 to H1 when heme and tryptophan were supplied. Thus, the purified enzyme, which was provisionally referred to as prostaglandin endoperoxide synthetase, exhibited two enzyme activities: the synthesis of prostaglandin G1 and its conversion to prostaglandin H1. Either free or protein-bound heme was required for both reactions, and only protoheme was active. Tryptophan stimulated the conversion of prostaglandin G1 to H1, and this stimulatory effect was also observed with various other aromatic compounds. Indomethacin and aspirin inhibited prostaglandin G1 synthesis, but not the other steps of prostaglandin biosynthesis.