Abstract Reactive oxygen species generated in living cells during normal cellular metabolism and in response to exogenous inducers cause the formation of oxidatively damaged bases in DNA. These oxidized base lesions are primarily removed by the base excision repair (BER) pathway. A BER enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol, from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against thymine glycol by empty-vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced mRNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. Next, we found two novel genetic polymorphisms, i.e., c. −163C>G and c. −241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. Next, all the coding exons of the NTH1 gene and their boundary regions in DNAs of 23 Japanese patients with gastric cancer was searched for genetic polymorphisms by direct sequencing. A novel variant, c. −31A>G, and a single nucleotide polymorphism, c.98G>A (Arg33Lys, rs2302172) were detected at an allele frequency of 2.2%, and 4.3%, respectively. However, the c. −31A>G polymorphism did not affect reduction of promoter activity. We are going to reveal functional characterization of c.98G>A and to examine excisional repair activity against new substrates, such as oligonucleotide containing etheno adducts. Our data suggested that downregulation of NTH1 expression, abnormal localization of NTH1 and the missense polymorphism may be involved in the pathogenesis of a subset of gastric cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2802.