Nitric oxide production is increased in the intestine in sepsis and may be a key component of sepsis-induced intestinal damage. Evidence suggests that iNOS activity increases along the crypt-villus axis of differentiating intestinal epithelium. We have performed an immunohistochemical analysis of iNOS expression in the rat ileum using indirect immunofluorescent microscopy. The effects of endotoxin (lipopolysaccharide; LPS) injection was examined in rats at three developmental ages: newborn (4 days postnatal), adolescent (3 wks, 140 gm) and adult (6 wks, 325 gm). Rats were injected with LPS, 5 mg per kg body weight, i.p. Ileum was removed after 3 hr, embedded, sectioned and fixed with acetone. Sections were stained with mouse anti-human iNOS and secondary antibody conjugated to florescein isothiocyanate (FITC). Following endotoxin exposure, positive and specific (i.e., primary antibody-dependent) immunostaining for iNOS was found in the mucosal layer of the ileum from adolescent and adult rats. Staining was most intense in epithelial cells at the villous tips. Little or no staining was seen in other cells. Expression was absent in non-LPS treated animals. In contrast, LPS-injected 4 day old rats showed a minimal level of iNOS immunoreactivity. No crypt to villus iNOS gradient was apparent. As was the case for older rats, non-injected 4 day old rats showed no iNOS staining. These data indicate that, in older rats: (1) Ileal iNOS expression is undetectable in the basal state and is markedly induced within 3 hr after LPS injection; (2) Ileal iNOS, once induced, is confined to epithelial cells; (3) Expression shows an increasing gradient from crypt to villus. Similar LPS-mediated induction of iNOS was not observed in newborn rat pups. This may be important for studies on the ontogeny of the pathophysiology of sepsis.