Oral leukoplakia is a premalignant lesion associated with development of oral cancer. To clarify the mechanism of development of oral carcinogenesis from leukoplakia, we examined DNA damage in oral epithelium of biopsy specimens of patients with leukoplakia by immunohistochemical methods. Histological changes, such as epithelial dysplasia and infiltration of inflammatory cells were observed in oral tissues of leukoplakia patients. A double immunofluorescence labeling study demonstrated that the accumulation of mutagenic 8-nitroguanine, an indicator of nitrative DNA damage, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, was apparently observed in the oral epithelium of patients with leukoplakia, whereas little or no immunoreactivity was observed in normal oral mucosa. Expression of inducible nitric oxide synthase (iNOS) was also observed in oral epithelium of leukoplakia patients. Immunoreactivity of 3-nitrotyrosine, an indicator of nitrative stress, was observed in oral epithelial cells and colocalized with 8-nitroguanine. Moreover, proliferating cell nuclear antigen and p53 were expressed in 8-nitroguanine-positive epithelial cells in the basal layer. These results suggest that iNOS-mediated nitrative stress contributes to development of oral carcinogenesis from leukoplakia through DNA damage as well as oxidative stress.