In addition to its role in absorbing nutrients, the intestinal mucosa provides an important barrier against toxins and bacteria in the bowel lumen. This study evaluated changes in rat jejunal permeability and histology after total parenteral nutrition (TPN) or TPN supplemented with glutamine. Lactulose and mannitol were used to measure jejunal permeability, and fixed stained histologic specimens were used to measure mucosal dimensions. After the insertion of central venous catheters, 18 male rats were randomly divided into three groups: CHOW, saline infusion with a standard laboratory rat diet ad libitum; TPN; and GLN, 2% L-glutamine-supplemented TPN. The TPN and GLN groups received isocaloric, isovolumic, and isonitrogenous feedings. After 7 days of infusion, a laparotomy was performed, and lactulose and mannitol were instilled into the lumen of a 25-cm ligated segment of jejunum. Urine was collected for 5 hours and assayed for lactulose, mannitol, and creatinine. The jejunum was harvested, and wet weight, villus height, mucosal thickness, and villus width were measured. Intestinal permeability to lactulose and the lactulose to mannitol ratio significantly increased after TPN compared with CHOW, and these effects were prevented with the addition of glutamine to the TPN solution. Jejunal villus height and mucosal thickness significantly decreased following TPN but were not significantly different from CHOW when glutamine was added to the TPN solution. These data suggest that TPN was associated with increased jejunal permeability and that glutamine, when added to the TPN solution, prevented this effect. In addition, glutamine reduced TPN-associated atrophy of the jejunum.