Elevated levels of ammonia in blood and brain result in derangement of cerebral function. Recently, lipid peroxidation and oxidative stress have been implicated in ammonia neurotoxicity. Because ammonia is primarily detoxified in astrocytes, we postulated that pathophysiological concentrations of ammonia might induce free radical formation in these cells. To test this hypothesis, we examined the extent of free radical production in primary cultures of astrocytes that had been preloaded with the fluorescent dye 5- (and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA). DCFDA fluoresence was found to be increased in a dose-dependent manner when astrocytes were exposed to 1, 5, and 10 mM NH4Cl. This phenomenon was transitory; it peaked at 2.5 min after exposure and declined subsequently. By 2 hr after treatment, DCFDA fluorescence was below control level. Addition of catalase or superoxide dismutase to 5 mM NH4Cl-treated astrocytes reduced free radical formation. Pretreatment with 3 mM methionine sulfoximine, an inhibitor of glutamine synthetase, also suppressed free radical formation by 5 mM NH4Cl. The results of this study suggest that elevated concentrations of ammonia induce the formation of free radicals in astrocytes and that this process is associated with the synthesis of glutamine. We propose that astrocyte-derived free radicals may be responsible for some of the pathophysiological changes associated with hyperammonemic conditions. J. Neurosci. Res. 66:282–288, 2001. © 2001 Wiley-Liss, Inc.