Hepatic encephalopathy (HE) is a major neurological complication that occurs in the setting of severe liver failure. Ammonia is a key neurotoxin implicated in this condition, and astrocytes are the principal neural cells histopathologically and functionally affected. Although the mechanism by which ammonia causes astrocyte dysfunction is incompletely understood, glutamine, a by-product of ammonia metabolism, has been strongly implicated in many of the deleterious effects of ammonia on astrocytes. Inhibiting mitochondrial glutamine hydrolysis in astrocytes mitigates many of the toxic effects of ammonia, suggesting the involvement of mitochondrial glutamine metabolism in the mechanism of ammonia neurotoxicity. To determine whether mitochondriaare indeed the organelle where glutamine exerts its toxic effects, we examined the effect of L-histidine, an inhibitor of mitochondrial glutamine transport, on ammonia-mediated astrocyte defects. Treatment of cultured astrocytes with L-histidine completely blocked or significantly attenuated ammonia-induced reactive oxygen species production, cell swelling, mitochondrial permeability transition, and loss of ATP. These findings implicate mitochondrial glutamine transport in the mechanism of ammonia neurotoxicity. © 2007 Wiley-Liss, Inc.