Prostaglandin synthetase has the ability to catalyze the metabolism of paracetamol to a reactive metabolite, which binds to protein and reduced glutathione (GSH). This was demonstrated with microsomes isolated from both sheep seminal vesicles (SSV) and rabbit kidney medulla. The activation of paracetamol occurred through cooxygenation during prostaglandin biosynthesis, the peroxidase activity of this enzyme being responsible for the reaction. In addition to being metabolized, paracetamol also stimulated the rate of prostaglandin biosynthesis probably by serving as a potent hydrogen donor. The metabolism of paracetamol to a reactive metabolite most likely involved the formation of a paracetamol radical species. This was indicated by an inhibitory effect of the antioxidant, butylated hydroxyanisole, as well as by a very rapid oxidation of GSH during the course of the prostaglandin synthetase catalyzed reaction. Whether this paracetamol radical is further oxidized to the quinone imine prior to reacting with GSH or protein, remains to be established. The ultimate reactive metabolite is evidently the same as that formed with liver microsomes and NADPH since the glutathione conjugates were apparently identical. The rate of paracetamol activation by SSV microsomes was, however, more than 100 times that by liver microsomes and furthermore the apparent Km was considerably lower. Finally, N-OH paracetamol was shown to be activated by prostaglandin synthetase in the presence of arachidonic acid to a metabolite apparently different from that formed from paracetamol.