Many reports have demonstrated that thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are biomarkers for the response and prognosis of patients treated with 5-fluorouracil (5-FU)-based chemotherapy. A newly developed orally administered drug, fluoropyrimidine (S-1), has been developed with clinical efficacy when combined with an inhibitor of DPD. In this study, the relationship between immunoreactivity to TS and DPD in biopsy specimens and the effects of chemotherapy was investigated in 41 patients treated with S-1 therapy for advanced gastric cancer. Response rates were 54% (13/24) in TS(+) and 53% (9/17) in TS(-) patients (p=0.938), and those of DPD(+) and (-) patients were 61% (11/18) and 48% (11/23) (p=0.397), respectively. The median survival time of all the subjects was 253 days. There was no significant difference in median survival time between TS(+) patients (284 days) and (-) patients (189 days: p=0.670). The 18 DPD(+) patients had median survival times slightly longer (338 days) than the 23 patients with DPD(-) (207 days: p=0.206). This study indicates that S-1 may be effective in the treatment of gastric cancer patients, regardless of intratumoral TS and DPD immunoreactivity status. Further studies are needed to confirm these results with larger numbers of patients.