At antipyretic and analgesic doses, salicylate has no antiplatelet or anti-inflammatory effects, unlike typical inhibitors of the prostaglandin H synthases (PGHSs). We demonstrated that salicylate inhibits PGHS-1 and -2 with a potency inversely related to ambient hydroperoxide concentrations. Salicylate inhibition of PGHS-1 was prevented by 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Increasing the production of prostaglandin G2 (the peroxide product of PGHS-cyclooxygenase activity) by elevating the concentration of either enzyme or substrate reversed inhibition. Using analogs of benzoic acid differing only at the hydroxyl position, we revealed the importance of this moiety to salicylate's inhibitory mechanism. Unlike typical phenolic inhibitors, e.g., acetaminophen, salicylate was ineffective as a reducing cosubstrate for PGHS-peroxidase activity, implicating the cyclooxygenase site as its putative target. PGHS-cyclooxygenase activity depends upon the oxidation of an active site tyrosine by electron transfer to the oxidized ferriprotoporphyrin of the peroxidase. The PGHS-1 apoenzyme reconstituted with manganese protoporphyrin instead of iron protoporphyrin has very little peroxidase activity. 12-HPETE does not prevent the inhibition of Mn-PGHS-1 by salicylate, indicating that reversal of salicylate inhibition by hydroperoxides depends upon electron transfer between the cyclooxygenase and peroxidase active sites. These results are consistent with an inhibitory action of salicylate at the PGHS-cyclooxygenase site that is dependent on the PGHS-peroxidase activity.