Abstract Infusion of total parenteral nutrition (TPN) with excess carbohydrate calories leads to hepatic steatosis in rats and is associated with an elevated portal insulin/glucagon molar ratio. Previously we have shown that adding glucagon to TPN prevents and reverses hepatic steatosis in rats, possibly by increasing hepatic lipid export. It has been reported that steatosis is eliminated in rats by the addition of l-glutamine to TPN. In this study, we examined the effect of glutamine on portal insulin and glucagon levels and the development of hepatic steatosis. Adult rats ( n = 19) received internal jugular catheters: Group 1 ( n = 6), saline (3 cc/hr) and chow ad libitum; Group 2 ( n = 7), 25% dextrose base TPN; Group 3 ( n = 6), 25% dextrose base TPN with 2% glutamine. The infusion rate of TPN was 1.2 cc/100 g body wt/hr. Daily nitrogen balance was determined and at 7 days, portal venous blood was drawn for insulin and glucagon radioimmunoassay, livers were removed for histology and lipid content determination, and the small intestines were removed for mucosal protein and DNA content determination. Panlobular vacuolization of the hepatocytes was noted on histology in Group 2 (TPN) while Group 1 (chow) and Group 3 (TPN + glutamine) showed normal liver morphology. Hepatic lipid content was significantly elevated in Group 2 ( P < 0.05). The portal insulin/glucagon molar ratio was increased because of excessive portal venous insulin in Group 2 (TPN). In contrast, portal glucagon was significantly elevated while the insulin/glucagon ratio and hepatic lipid content did not increase above control levels in the glutamine-supplemented Group 3 rats. The results suggest that addition of l-glutamine to hypertonic dextrose TPN prevents the development of hepatic steatosis, possibly by stimulating glucagon secretion and thus lowering the portal insulin/glucagon ratio and increasing hepatic lipid export.