The microsomal fraction prepared from canine myocardium was found to contain an active prostaglandin synthetase (average, 10 mμ mol PGE2/mg protein/min) which was capable of forming PGE2 from arachidonic acid in the presence of reduced glutathione and cofactors such as l-adrenaline, hydroquinone, and p-aminophenol. Neither aspirin nor phospholipase A, which have been reported to affect prostaglandin synthesis in other tissues, influences the activity of myocardial prostaglandin synthetase.