The microsomal fraction of bovine vesicular gland catalyzed the conversion of eicosapolyenoic acids exclusively to prostaglandin E in the presence of reduced glutathione, while hydrox fatty acids, prostaglandins D and F, decreased to a negligible level. After solubilizing the microsomal fraction with cutscum, the prostaglandin synthetase activity was purified 11-fold by batchwise absorption and elution of the enzyme activity from DEAE-cellulose. This partially purified enzyme fraction did not respond to reduced glutathione in promoting prostaglandin E formation at the expense of other products. A number of glutathione analogs were examined, but none of these was as effective as reduced glutathione. Dithiol complexes of Cu-2+, Ni-2+, and Zn-2+ exerted pronounced effects on relative amounts of the different prostaglandins biosynthesized. Both the Cu-2+-dithiothreitol (2:1) complex and stannous chloride markedly enhanced prostaglandin F synthesis at the expense of prostaglandin D and prostaglandin E. The following reagents chemically reduced the endoperoxide in ascaridole to p-menth-2-ene-cis-1,4-diol: Cu-2+0dithiothreitol, Cu-2+-epinephrine, and stannous chloride. It is concluded that the enhancement of prostaglandin F formation caused by copper-dithiols and L-epinephrine is due to nonenzymatic reduction of prostaglandin G or prostaglandin H.