Porphyromonas gingivalis is a primary etiological agent of generalized severe periodontitis, and emerging data suggest the importance of reactive oxygen and nitrogen species in periodontal tissue damage, as well as in microbial killing. Since nitric oxide (NO) released from inducible NO synthase (iNOS) has been shown to possess immunomodulatory, cytotoxic, and antibacterial effects in experimental models, we challenged iNOS-deficient (iNOS^−/−) mice with P. gingivalis by using a subcutaneous chamber model to study the specific contribution of NO to host defense during P. gingivalis infection. iNOS^−/− mice inoculated with P. gingivalis developed skin lesions and chamber rejection with higher frequency and to a greater degree than similarly challenged C57BL/6 wild-type (WT) mice. Chamber fluid from iNOS^−/− mice possessed significantly more P. gingivalis than that of WT mice. The immunoglobulin G responses to P. gingivalis in serum was similar in WT and iNOS^−/− mice, and the inductions of tumor necrosis factor alpha, interleukin-1β and interleukin-6, and prostaglandin E₂ were comparable between the two mouse strains. Although no differences in total leukocyte counts in chamber fluids were observed between iNOS^−/− and WT mice, the percentage of dead polymorphonuclear leukocytes (PMNs) was significantly greater in iNOS^−/− mouse chamber fluids than that of WT samples. Interestingly, casein-elicited PMNs from iNOS^−/− mice released more superoxide than did WT PMNs when stimulated with P. gingivalis. These results indicate that modulation of superoxide levels is a mechanism by which NO influences PMN function and that NO is an important element of the host defense against P. gingivalis.