Nitric oxide (NO) is implicated in the pathophysiology of intestinal inflammation. Intestinal mast cells may amplify inflammatory response and mucosal injury in inflammatory bowel disease. Our aim was to examine the role of NO and intestinal mast cells by investigating the effects of NO synthase (NOS) inhibitors and a mast cell stabilizer during induction of dextran sulfate sodium (DSS) colitis. Colitis was induced by 4% DSS in drinking water, in rats pretreated with L-NAME or aminoguanidine. In another set of experiments, we investigated the effect of ketotifen in this setting. Inhibition of NO by L-NAME worsened DSS-induced inflammation, however, aminoguanidine had no effect. On the other hand, ketotifen abolished the deleterious effects of L-NAME on colonic inflammation, suggesting that hyperactivation of mast cells by NOS inhibition amplifies mucosal injury induced by DSS. Our results suggest that constitutive isoforms of NOS prevent mast cell activation.