The activities of rat brain prostaglandin D synthetase and swine brain postaglandin D 2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC 50 values of this acid were 80 μM for prostaglandin D synthetase and 7 μM for prostaglandin D 2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC 50 values of these derivatives were more than 200 μM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC 50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 μM for prostaglndin D synthetase, and 20, 10 8.5 and 7 μM for prostaglandin D 2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC 50 values of 40 μM for prostaglandin D synthetase and 3.9 μM for prostaglandin D 2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC 50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 μM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 μM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 μM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 μM).