Aims: Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE). Neurotoxic effects of ammonia induce numerous metabolic alterations such as increased brain lactate (Lac) and glutamine (Gln) levels. Brain edema is present in patients with chronic liver failure however the pathophysiological mechanisms are not clearly understood. The present study investigates the role of brain ammonia, Lac and Gln in pathogenesis of brain edema in 2 different rat models of chronic liver failure/HE; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: Ammonia (commercial kit), brain edema (specific gravimetric technique) Lac (Amplex Red fluorescence) and Gln (1H nuclear magnetic spectroscopy) were measured in brain tissue of PCA and BDL rats vs sham operated controls. De novo synthesis of Lac and Gln was assessed in brain tissue by 13C nuclear magnetic resonance spectroscopy, after injecting PCA and BDL rats along with their respective sham operated controls with [U-13C] glucose (500 mg/kg, i.p) 30 minutes before sacrifice. Lac, Gln and brain edema were also investigated in BDL rats at 2, 4 and 6 weeks after surgery and after reducing ammonia levels with AST-120 (spherical carbon adsorbent), at 1g/kg/day for 6 weeks. Results: Hyperammonemia developed in both PCA (177.3±8.9μM vs sham: 52.1±11.3μM; p<0.0001) and BDL (155.0±8.9μM vs sham: 41.0±11.2μM; p<0.0001); with no significant difference between the groups. An increase in brain water content was observed in rats with BDL (81.88±0.12% vs sham: 81.13±0.15%, p<0.05), as well as total Lac levels; whereas no significant change in brain water and total Lac was found in PCA. Gln levels significantly increased in PCA rats and BDL rats vs respective sham-operated controls. A significant increase in de novo synthesis of 13C Lac and 13C Gln from 13C glucose was found in BDL rats, whereas in PCA rats only de novo synthesis of 13C Gln increased. In addition, total Lac levels were increased in BDL rats before the apparition of brain edema. Following AST-120 treatment, Lac levels and brain edema significantly decreased, while Gln levels remained unchanged. Conclusions: BDL and PCA rats both presented with similar levels of hyperammonemia and Gln, as well as increased de novo synthesis of Gln from glucose. However only BDL rats demonstrated an increase in brain water and Lac as well as increased de novo synthesis of Lac from glucose. Our findings suggest increased brain Lac, not increased brain Gln, is associated with brain edema. We conclude that impaired Lac metabolism may contribute to the pathogenesis of brain edema in HE.