OBJECTIVE: To determine effects of glutamine-supplemented and glutamine-free amino acid-based purified diets, compared with a dry expanded diet, on intestinal structure and function in a model that used cats with methotrexate-induced enteritis. ANIMALS: 18 adult specific-pathogen-free cats. PROCEDURE: 12 cats were given intragastric feedings of an amino acid-based purified diet supplemented with glutamine (7% [wt:wt]) or an isonitrogenous amount of glycine and alanine; 6 cats consumed a dry expanded diet. After 21 days, cats received methotrexate (MTX; 11 mg/kg of body weight, IV). Intestinal permeability testing was performed immediately before and 66 hours after MTX administration. Celiotomy was performed 72 hours after MTX administration for aseptic removal of mesenteric lymph nodes, collection of full-thickness intestinal biopsy specimens, determination of intestinal cellular proliferation, and collection of aortic and portal venous blood samples for determination of arteriovenous amino acid concentrations across the intestine. RESULTS: Administration of MTX was associated with severe enterotoxicosis manifested as diarrhea (8/12 cats), vomiting (12/12), and positive results for bacterial culture of mesenteric lymph nodes (12/12) in cats receiving the purified diets, independent of glutamine supplementation. Diet did not affect villus tip length and villus surface area in the small intestine or cellular proliferation. Administration of MTX was associated with significantly increased intestinal permeability, which was not attenuated by glutamine supplementation. CONCLUSIONS: Feeding of a glutamine-supplemented amino acid-based purified diet was unable to preserve intestinal function in cats with MTX-induced enteritis. CLINICAL RELEVANCE: Intestinal morphologic alterations correlate poorly with intestinal function as measured by means of bacterial translocation and intestinal permeability.