Nitric oxide (NO) produced by the NO synthase (NOS), a family of enzymes such as inducible NOS (iNOS), has been suggested to play an important role in tumor bioloty. We immunohistochemically examined iNOS and p53 protein expression in 105 patients with esophageal squamous cell carcinoma (ESCC). Direct sequence analysis for the p53 gene was performed in 51 of 105 tumors. In total, 56 of 105 (53.3%) tumors exhibited intracytoplasmic staining for anti-iNOS antibody, including 17 (16.2%) cases of homogeneous and intense immunostaining (++) and 39 (37.1%) of heterogeneous staining (+). Of 62 p53 protein-positive tumors, 40 (63.5%) were positive for iNOS, and of 43 p53 protein-negative tumors, 27 (62.8%) were negative for iNOS. Of 34 iNOS-positive tumors, 23 (67.6%) carried a p53 gene mutation, and of 17 iNOS-negative tumors, 12 (70.6%) had wild-type p53 gene. There was a significant relationship between iNOS immunoreactivity and p53 protein overexpression (p = 0.0058) as well as p53 mutation frequency (p = 0.0163). No association was found between iNOS immunoreactivity and p53 mutation type, any clinicopathological factor and patient prognosis. Our in vivo findings suggest that iNOS activity might be associated with p53 alteration and contribute to tumorigenesis in ESCC.