Disturbed body nitrogen homeostasis due to impaired hepatic urea synthesis leads to an alteration in inter-organ ammonia trafficking, resulting in hyperammonemia. Glutamine (Gln) synthase is the alternative pathway for ammonia detoxification. Gln taken up by several organs is split by the intramitochondrial phosphate-activated enzyme glutaminase (PAG) into glutamate (Glu) and ammonia. In cirrhotic patients with portosystemic intrahepatic shunt, the main source of systemic hyperammonemia is the small intestine, and ammonia derives mainly from Gln deamidation. Recently, PAG has been found increased in cirrhotics showing minimal hepatic encephalopathy and, therefore, could be implicated in the production of systemic hyperammonemia in these patients. Intestinal PAG activity correlates with psychometric test and magnetic resonance spectroscopy findings. Moreover, nitric oxide and tumor necrosis factor seem to be the major factors regulating intestinal ammonia production in cirrhotics. In the brain, PAG localized into the astrocytes is responsible for ammonia and free-radical production. The blockade of PAG, using 6-oxo-5-norleucine, avoids the toxic effects of Gln accumulation in the brain. These data support an important role for intestinal and brain glutaminase in the pathogenesis of hepatic encephalopathy and could be a new target for future therapies.