Glutamine synthetase (GS) in the liver is restricted to a small perivenous hepatocyte population and plays an important role in the scavenging of ammonia that has escaped the periportal urea-synthesizing compartment. We examined the effect of a single intraperitoneal injection of lipopolysaccharide (LPS) in vivo on glutamine synthesis in rat liver. LPS injection induced expression of inducible nitric oxide synthase, which was maximal after 6 to 12 hours but returned toward control levels within 24 hours. Twenty-four hours after LPS injection, an approximately fivefold increase in tyrosine-nitrated proteins in liver was found, and GS protein expression was decreased by approximately 20%, whereas GS activity was lowered by 40% to 50%. GS was found to be tyrosine-nitrated in response to LPS, and immunodepletion of tyrosine-nitrated proteins decreased GS protein by approximately 50% but had no effect on GS activity. Together with the finding via mass spectrometry that peroxynitrite-induced inactivation of purified GS is associated with nitration of the active site tyrosine residue, our data suggest that tyrosine nitration critically contributes to inactivation of the enzyme. In line with GS inactivation, glutamine synthesis from ammonia (0.3 mmol/L) in perfused livers from 24-hour LPS-treated rats was decreased by approximately 50%, whereas urea synthesis was not significantly affected. In conclusion, LPS impairs hepatic ammonia detoxification by both downregulation of GS and its inactivation because of tyrosine nitration. The resulting defect of perivenous scavenger cell function with regard to ammonia elimination may contribute to sepsis-induced development of hyperammonemia in patients who have cirrhosis.