Nitric oxide (NO), an important regulatory molecule for immune response and cytotoxicity, is endogenously generated from L-arginine by NO synthase (NOS). One mechanism for NO-induced cytotoxicity is through its interaction with superoxide to produce peroxynitrite, which causes DNA damage. Three distinct isoforms of NOS have been isolated and represent the products of three different genes. The inducible form, inducible nitric oxide synthase (iNOS), is a mediator of inflammation and a regulator of epithelial cell growth. Upregulation of iNOS has been linked to epithelial tumorigenesis in various human and animal tissues. In the current investigation, normal esophagus and N-nitrosomethylbenzylamine (NMBA)-induced preneoplastic and papillomatous lesions of the rat esophagus were characterized for expression of iNOS. F344 rats were injected subcutaneously with NMBA (0.5 mg/kg body weight) three times per week for 5 wk. At 3, 6, 9, 12, 15, 18, 21, 24, 30, and 36 wk following initiation of NMBA treatment, esophagi were collected from 12 untreated and 12 NMBA treated animals. Results of reverse transcription (RT)-polymerase chain reaction (PCR) and immunohistochemistry demonstrated a correlation between the upregulation of iNOS and neoplastic progression in the rat esophagus. The expression of iNOS mRNA in preneoplastic tissues and papillomas was significantly elevated when compared to normal tissues. Immunohistochemical analysis showed more extensive cytoplasmic staining of iNOS protein in preneoplastic tissues and papillomas than in normal tissues. Our data suggest, therefore, that the production of iNOS by the epithelium of the esophagus is associated with the development of NMBA-induced esophageal tumorigenesis in rats.