The liver plays a major role in regulating ammonia levels in the blood . Therefore, in liver disease, the loss of hepatic function leads to hyperammonemia and consequently hepatic encephalopathy (HE) . Ammonia-lowering strategies remain the mainstay therapeutic strategy for HE . Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain . Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease . GS is expressed in liver, muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB . Methods: Using brain primary microvascular endothelial cells (ECs) and isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats, the presence of GS was assessed using rtPCR, western blot (brain used as positive control), immunohistochemistry (co-localized with endothelial cell marker caveolin-1) . GS activity was also assessed . In addition, to understand the effect of liver disease on GS, ECs were exposed to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) and sham-operated controls, as well as ammonia chloride (1mM) and oxidative stress (10μM of hydrogen peroxide) . Results: Immunohistochemically, GS was detected in ECs and in vessels of brain from naïve rats . Furthermore, ECs expressed GS mRNA, protein and activity . However, expression of GS was lower compared to brain lysate control samples (p<0 .05) . GS expression in CMV showed similar results to brain but GS activity was less (p<0 .05) . When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0 .05) . However, when exposed to plasma from BDL rats or oxidative stress, GS activity was significantly reduced when compared to controls (p<0 .01) . Conclusion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro . Interestingly, ammonia stimulates GS in endothelial cells, whereas oxidative stress inhibits GS activity . Furthermore, GS activity is decreased in the presence of plasma from cirrhotic rats, possibly due to the presence of systemic oxidative stress in BDL rats. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may increase the risk of HE . We anticipate that upregulating GS in ECs of the BBB could become a new therapeutic target for HE.