Significant atrophy and impaired absorption occur in the heterotopically transplanted small intestinal isograft, and these deficits are corrected when the preferred fuel of the enterocyte, glutamine (Gln), is supplemented to total parenteral nutrition (TPN). In the orthotopic small bowel isograft, this study determined whether Gln-enriched TPN enhanced mucosal structure and function, and decreased bacterial translocation to mesenteric lymph nodes (MLN). Seventeen adult Lewis rats received orthotopic jejunal isografts and central venous catheters for TPN. Rats received either TPN with 2% Gln or the same TPN with isonitrogenous balanced nonessential amino acids for 10 days. Eight normal, chow-fed rats served as baseline controls. Mucosal villous height, surface area, crypt depth, weight, protein and DNA contents, brush border enzymes, 14C glucose absorption, and bacterial translocation to MLN were evaluated in both the graft and host jejunum and the control animals. Gln-enriched TPN significantly increased mucosal villous height (P < 0.01), surface area (P < 0.01), and glucose absorption (P < 0.01), and it reduced bacterial translocation (P < 0.05) when compared with the non-Gln TPN group. For most study variables, there were no significant differences between Gln-enriched TPN or baseline and between the graft and host jejunum for Gln- and non-Gln-supplemented animals. There were no significant differences in DNA content and brush border enzymes among groups. These results indicate that Gln-enriched TPN improves mucosal structure and glucose absorption and reduces bacterial translocation to MLN in the orthotopic small bowel isograft.