Peroxynitrite (ONOO^-), the reaction product of the interaction between superoxide (O₂·^-) and nitric oxide (·NO), is a potent proinflammatory and cytotoxic nitrooxidative species. Its role as a mediator of hyperalgesia (clinically defined as an augmented sensitivity to painful stimuli) is not known. In light of the known proinflammatory properties of ONOO^-, our study addressed its potential involvement in the development of hyperalgesia associated with tissue damage and inflammation. Intraplantar injection in rats of the ONOO^- precursor O₂·^- (1 µM) led to the development of thermal hyperalgesia associated with a profound localized inflammatory response. Both events were blocked by L-NAME (N^G-nitro-L-arginine methyl ester, 3–30 mg/kg), a nitric oxide synthase inhibitor, or by FeTM-4-PyP⁵⁺ [Fe(III)5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, 3–30 mg/kg], an ONOO^- decomposition catalyst. These results suggested that locally synthesized ONOO^- produced in situ by O₂·^- and ·NO is key in the development of inflammatory hyperalgesia. The direct link between ONOO^- and hyperalgesia was further supported by demonstrating that intraplantar injection of soluble ONOO^- itself (1 µM) similarly led to inflammatory hyperalgesia. ONOO^- generated by the interaction between exogenous administration of O₂·^- and endogenous ·NO, or provided by direct injection of ONOO^-, activated the transcription factor NF-B in paw tissues, enhancing expression of the inducible but not the constitutive cyclooxygenase enzyme (COX-2 and COX-1, respectively). ONOO^--mediated hyperalgesia was blocked in a dose-dependent manner by intraperitoneal injections of indomethacin (10 mg/kg), a nonselective COX-1/COX-2 inhibitor, or NS398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; 10 mg/kg] a selective COX-2 inhibitor, as well as by an anti-prostaglandin (PG) E₂ antibody (200 µg). In another established model of inflammation-related hyperalgesia by intraplantar injection of carrageenan in rats, inhibition of ONOO^- with FeTM-4-PyP⁵⁺ (3–30 mg/kg) inhibited the development of hyperalgesia and the release of PGE₂ in paw tissue exudates. Furthermore, FeTM-4-PyP⁵⁺ synergized with indomethacin and NS397 (1–10 mg/kg) to block both hyperalgesia and edema. Taken together, these data show for the first time that ONOO^- is a potent mediator of inflammation-derived hyperalgesia operating via the COX-to-PGE₂ pathway. These results provide a pharmacological rationale for the development of inhibitors of peroxynitrite biosynthesis as novel nonnarcotic analgesics. The broad implications of our study are that dual inhibition of both ONOO^- formation and COX activity may provide an alternative therapeutic approach to the management of pain: effective analgesia with reduced side-effects typically associated with the use of COX inhibitors.—Ndengele, M. N., Cuzzocrea, S., Esposito, E., Mazzon, E., Di Paola, R. Matuschak, G. M., Salvemini, D. Cyclooxygenases 1 and 2 contribute to peroxynitrite-mediated inflammatory pain hypersensitivity.