Partially purified glutamine synthetase from rat liver underwent rapid inactivation upon incubation with NADH and benzyl viologen, under aerobic conditions. This in vitro inactivation was prevented by catalase or chelating-agents, which suggests that hydrogen peroxide and metal ions are involved in the process. Similar inactivation was observed when the rat liver glutamine synthetase was preincubated, under anaerobic conditions, with NADH and benzyl viologen, and hydrogen peroxide was added to the reaction mixture. A radical scavenger, histidine, partially prevents the inactivation, while hydrogen peroxide shows a low inactivation capacity when incubated without NADH. Furthermore, the fact that the inactivation can also be catalyzed by a model consisting of ferrous ions and hydrogen peroxide leads to the conclusion that hydroxyl radicals, or something with similar reactivity, are most likely produced through a Fenton reaction.