We investigated the pathogenic mechanism of indomethacin-induced small intestinal lesions, in relation to nitric oxide (NO), superoxide radicals and mast cells. Rats received indomethacin (1-6 mg/kg) s.c. once daily for 3 days, and the small intestine was examined for lesions 24 hr after the final administration of indomethacin. Indomethacin caused hemorrhagic lesions in the small intestine, mostly in the jejunum and ileum, in dose- and time-dependent manners, with concomitant increase of mucosal microvascular permeability. This treatment also caused an increase of inducible NO synthase (iNOS) activity with the expression of its mRNA, myeloperoxidase (MPO) activity as well as thiobarbituric acid reactants (TRBAS) in the mucosa, and the changes in iNOS activity preceded those in MPO activity and TRBAS as well as lesion development. These lesions induced by indomethacin were prevented by aminoguanidine (a selective inhibitor of iNOS), dexamethasone (an inhibitor of iNOS mRNA transcription), allopurinol (a xanthine oxidase inhibitor), hydroxyurea (a neutrophil reducing agent) and FR167653 (an inhibitor of interleukin-1/tumor necrosis factor-alpha production) as well as 16,16-dimethyl prostaglandin E2ń. Likewise, the severity of these lesions was also reduced by mast cell stabilizers FPL-52694 and disodium cromoglycate and a lipoxygenase inhibitor TMK-688, but not affected by tripelennamine (a histamine H1-receptor antagonist) or methysergide (a serotonin receptor antagonist). These results suggest that: 1) the pathogenic mechanism of indomethacin-induced small intestinal lesions involves superoxide radicals as well as NO produced by iNOS, 2) the deleterious effect of NO may be accounted for by the cytotoxic action of peroxynitrite, produced from NO in the presence of superoxide radicals, and 3) the mast cells may also be involved in the process of small intestinal ulceration, although the mediator responsible remains undefined.