Cyclic prostaglandin endoperoxides prostaglandin G2 and H2 are intermediates formed in the biosynthesis of prostaglandins from arachidonic acid. These endoperoxides can be converted chemically or enzymatically to prostaglandins E2, D2 and F2alpha. The effects of several reducing compounds on the chemical and enzymatic transformations of prostaglandins G2 and H2 were studied in order to determine the possible existence of two alternative enzymatic pathways for the conversion of prostaglandin G2 to prostaglandins. The chemical transformation of prostaglandin H2 to prostaglandins by cleavage of the 9,11-cycloendoperoxide ring was unaffected by the presence of reduced glutathione, heme or tryptophan while hydroquinone and mercaptoethanol promoted the chemical reduction to prostaglandin F2alpha. In contrast the enzymatic transformation of prostaglandin H2 to prostaglandins by a solubilized prostaglandin synthetase from sheep vesicular gland was unaffected by hydroquinone or mercaptoethanol, but was markedly stimulated by reduced glutathione to yield mainly prostaglandin E2. Prostaglandin G2 transformation to prostaglandins involves cleavage of the 9,11-endoperoxide ring and a reduction of the 15-hydroperoxy group. The chemical reduction in buffer in the 15-hydroperoxy group is not affected by the presence of reduced glutathione, hydroquinone, heme or tryptophan. In contrast, the enzymatic reduction of the 15-hydroperoxy group is catalyzed by the solubilized prostaglandin synthetase and is further stimulated by reduced glutathione and hydroquinone. Results are presented which indicate that the enzymatic conversion of prostaglandin G2 to prostaglandins can proceed via two alternative pathways, one involving the intermediate formation of prostaglandin H2 and the other the formation of 15-hydroperoxy prostaglandins. The latter pathway appears to be the major pathway for the enzymatic conversion of prostaglandin G2 to prostaglandins.