Prostaglandin E(2) synthase (PGE synthase) is one of the membrane-associated proteins in the eicosanoid and glutathione metabolism (MAPEG) family of microsomal enzymes and constitutes a novel inducible enzyme involved in inflammation and pyretic responses. We report, using a reversed-phase HPLC assay for the production of tritiated prostaglandin E(2) (PGE(2)) by membranes from cells overexpressing human microsomal PGE synthase, that PGE synthase activity is inhibited effectively by 15-deoxy-Delta(12,14)-prostaglandin J(2) and arachidonic acid. The anti-inflammatory compound 15-deoxy-PGJ(2) was considerably more potent at inhibiting PGE synthase (IC(50)=0.3 microM) than the closely related PGJ(2) or Delta(12)-PGJ(2), or the reaction product PGE(2). Arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid inhibited PGE synthase with a similar potency (IC(50)=0.3 microM) and were more potent inhibitors than various fatty acid analogues. The present results on the inducible PGE synthase extend observations on the ability to bind arachidonic acid to another member of the MAPEG family, and also suggest a novel mechanism of action for the anti-inflammatory effects of DHA, EPA, and 15-deoxy-PGJ(2).