We have directly demonstrated in vivo that nitric oxide (NO) is produced in the ischemic rat brain. Using diethyldithiocarbamate and Fe as spin-trapping agents, NO spin adducts were detected by cryogenic electron paramagnetic resonance. The cerebral cortex which was exposed to focal ischemia or bilateral carotid artery occlusion generated an increased amount of spin-adducts of NO radicals (g = 2.039, a hyperfine coupling constant aN = 13 gauss). This signal disappeared by the preischemic administration of NG-nitro-L-arginine methylester, a NO synthase inhibitor.