Oral feeding of DL-difluoromethyl ornithine (DFMO) (2% in water ad libitum) for 14 days has no detectable effect on the small intestine of adult rats. Similar feeding of DFMO to weanling rat pups caused diarrhea in three to four days accompanied by a decrease in food consumption and body weight compared to age-matched controls. Significant decreases in small intestinal mucosal weight, total protein, DNA, enterokinase, leucine amino peptidase, sucrase, and maltase contents were observed in the DFMO-treated group four days after treatment. Extending the treatment to seven days led to a more severe reduction in these parameters. Villous atrophy of the mucosa was demonstrable by light microscopy and morphometric measurements. The mucosa of the DFMO-treated rat pups showed a reduction in total thickness and villous height but no change in crypt depth. A significant reduction in villus-crypt ratio was also seen. Changes in small intestinal mucosal parameters were not due to a decrease in food intake since pair-fed, age-matched rat pups showed no biochemical changes compared to control pups. DFMO-treated weanling rats showed less than 5% of ornithine decarboxylase (ODC) activity when compared to age-matched control animals. The effects observed on the small intestinal mucosa are presumably due to inhibition of ornithine decarboxylase activities by DFMO which prevents the proliferation, regeneration, and maturation of epithelial cells. The relative insensitivity of the adult rat small intestine to DFMO treatment suggests a lesser dependence of its intestinal mucosa to ODC activities.