Mice deficient in both inducible nitric oxide synthase (iNOS) and interleukin (IL)-10 (iNOS−/−/IL-10−/−) were created to examine the role of iNOS in spontaneously developing intestinal inflammation. IL-10−/−/iNOS−/−mice were compared with IL-10−/−(iNOS+/+) littermates over 6 mo. RT-PCR, Western blot analysis, and immunohistochemistry were performed to measure iNOS message and protein levels. Plasma nitrate/nitrite (NOx) levels were assessed by HPLC. Damage scores (macroscopic and microscopic) and granulocyte infiltration were assessed. At 3–4 wk, IL-10−/−and IL-10−/−/iNOS−/−mice had no signs of colonic inflammation or granulocyte infiltration. Plasma NOxlevels were not different from controls. By 3–4 mo, IL-10−/−mice had increased damage scores and granulocyte infiltration concurrent with increased mRNA and protein synthesis (restricted to the epithelium) for iNOS in intestinal tissues but not other tissues. Plasma NOxlevels increased fivefold. Interestingly, in the absence of iNOS induction or increased plasma NOx, iNOS−/−/IL-10−/−mice had damage and granulocyte infiltration equivalent to those observed in IL-10−/−littermates. These data suggest that iNOS does not impact on the development or severity of spontaneous chronic inflammation in IL-10-deficient mice.