Nitric oxide (NO) produced by constitutively expressed NO synthase (cNOS) plays an important role in maintaining the mucosal integrity of the small intestine, in collaboration with prostaglandins produced by cyclooxygenase (COX)-1. We examined whether intestinal damage is provoked in rats under inhibition of both cNOS and COX-2. The animals were given L-NAME (N(G)-nitro-L-arginine methyl ester), aminoguanidine, or rofecoxib, either alone or in combination, and killed 24 h later. Neither L-NAME nor aminoguanidine alone provoked damage in the small intestinal mucosa within 24 h, yet L-NAME produced damage in a L-arginine-sensitive manner when administered together with rofecoxib. L-NAME up-regulated the expression of COX-2 mRNA, and the prostaglandin E(2) (PGE(2)) content following the L-NAME administration significantly increased 12 h later, in both a rofecoxib- and a L-arginine-inhibitable manner. L-NAME enhanced intestinal motility, decreased mucus secretion, and increased the number of bacteria in the mucosa. The up-regulation of COX-2 expression and PGE(2) production by L-NAME was inhibited by prior administration of atropine, at a dose that inhibited the intestinal hypermotility. The intestinal lesions induced by L-NAME plus rofecoxib were prevented by pretreatment with ampicillin and aminoguanidine as well as atropine, indicating the involvement of bacteria, inducible nitric oxide synthase, and hypermotility in the pathogenesis. These results suggest that inhibition of both cNOS and COX-2 provokes intestinal damage, similar to inhibition of both COX-1 and COX-2. Inhibition of cNOS, similar to COX-1, up-regulates COX-2 expression, the process being associated with intestinal hypermotility and bacterial invasion, and this may be a key to the occurrence of intestinal damage associated with COX-2 inhibition.