To assess reactive oxygen species (ROS) production by detecting the fluorescent oxidation product, hydroethidine has been used extensively. The present study was undertaken to evaluate the potential of the hydroethidine derivative as a radiotracer to measure in vivo brain ROS production. [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]Hydromethidine) was synthesized, and evaluated using in vitro radical-induced oxidization and in vivo brain ROS production model. In vitro studies have indicated that [(3)H]Hydromethidine is converted to oxidized products by a superoxide radical (O(2)(•)-) and a hydroxyl radical (OH(•)-) but not hydrogen peroxide (H(2)O(2)). In vivo whole-body distribution study showed that [(3)H]Hydromethidine rapidly penetrated the brain and then was washed out in normal mice. Microinjection of sodium nitroprusside (SNP) into the brain was performed to produce ROS such as OH(•)- via Fenton reaction. A significant accumulation of radioactivity immediately after [(3)H]Hydromethidine injection was seen in the side of the brain treated with SNP (5 and 20 nmol) compared with that in the contralateral side. These results indicated that [(3)H]Hydromethidine freely penetrated into the brain where it was rapidly converted to oxidized forms, which were trapped there in response to the production of ROS. Thus, [(3)H]Hydromethidine should be useful as a radical trapping radiotracer in the brain.