Heme oxygenase activity was examined in the epithelial cells of the small intestine in male Sprague-Dawley rats. As with liver and spleen, the highest specific activity of this enzyme was found in the microsomal fraction of these cells. Substrate kinetics, analysis of cofactor requirements, and other biochemical characteristics suggested further similarities between heme oxygenase in the small intestine and liver. Enzyme activity was differentially localized longitudinally within the small intestine, with the highest specific activity occurring in the region approximately 15 to 30 cm beyond the pylorus. The effects of diet on the basal levels of heme oxygenase in the proximal small intestine were also examined. Although intestinal cytochrome P450-dependent monooxygenase activity, as determined by benzo[a]pyrene hydroxylase and 7-ethoxycoumarin O-deethylase, was greatly reduced (65-90%) in animals maintained on a semipurified control diet compared with standard cereal-based chow, there were no differences observed in heme oxygenase activity between the two dietary treatment groups. The activity of intestinal heme oxygenase could be increased, however, by oral treatment with several metal compounds that are known to affect hepatic heme metabolism when administered parenterally. The enzyme activity was also potently inhibited by tin (Sn4+) protoporphyrin administered orally or parenterally.