The effect of endotoxin on renal glutamine metabolism and ammoniagenesis was investigated in vivo in the rat to gain further insight into the altered glutamine flow that characterizes critical illness. Studies were done 15 hours following a single dose of Escherichia coli lipopolysaccharide (10 mg/kg). Renal blood flow and arterial glutamine concentration were similar in control and study rats, but the kidney switched from an organ of slight glutamine uptake in controls (129 ± 52 nmol/100 g of body weight per minute) to net release in the endotoxin-treated animals ( −273 ± 170 nmol/100 g of body weight per minute). Simultaneously, the specific activity of renal glutamine synthetase increased by almost 50% (374 ± 40 nmol/mg of protein per hour in rats given endotoxin vs 253 ± 12 nmol/mg of protein per hour in controls), while glutaminase was unchanged. Urinary ammonia excretion was reduced by 35% in the endotoxin-treated animals (47±6 μmol/12 h in endotoxin-treated animals vs 70±8 μmol/12 h in controls) despite a 10% fall in the arterial bicarbonate value. Endotoxin alters the net flux of glutamine across the kidney which appears to be partially regulated enzymatically. This may impair the kidneys' ability to maintain acid/base homeostasis. © 1991, American Medical Association. All rights reserved.