Inhibition of prostaglandins synthesis does not completely explain non-steroidal anti-inflammatory drug-induced spinal antinociception. Among other mediators, endocannabinoids are involved in pain modulation. Indomethacin-induced antinociception, in the formalin test performed in spinally microdialysed mice, was reversed by co-administration of the cannabinoid 1 (CB(1)) antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide (AM-251), but not by co-infusion of prostaglandin E(2). Indomethacin was ineffective in CB(1) knockout mice. AM-251 also reversed the indomethacin-induced antinociception in a test of inflammatory hyperalgesia to heat. Furthermore, during the formalin test, indomethacin lowered the levels of spinal nitric oxide (NO), which activates cellular reuptake and thus breakdown of endocannabinoids. The pronociceptive effect of an NO donor, 3-methyl-N-nitroso-sydnone-5-imine (RE-2047), was abolished by co-administration of the endocannabinoid transporter blocker N-(4-hydroxyphenyl) arachidonoyl amide (AM-404). Moreover, the antinociceptive activity of the NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), was reversed by AM-251. Thus we propose that at the spinal level, indomethacin induces a shift of arachidonic acid metabolism towards endocannabinoids synthesis secondary to cyclooxygenase inhibition. In addition, it lowers NO levels with subsequent higher levels of endocannabinoids.