We conducted a phase I/II study of irinotecan (CPT-11) combined with S-1, an oral dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine, in advanced gastric cancer (AGC). S-1 was administered orally at 80 mg/m2/day from day 1 to 14 of a 28-day cycle and CPT-11 was given intravenously on day 1 and 8 at initial dose of 70mg/m2/day, stepping up to 100mg/m2. The treatment was repeated every 4 weeks, unless disease progression was observed. In the phase I portion, the recommended dose of CPT-11 was determined as 80mg/m2. In the phase II portion, 42 patients were evaluated. The median treatment course was five (range: 1-13). The incidences of severe (grade 3-4) hematological and nonhematological toxicities were 19% and 10%, respectively, but all were manageable. The response rate was 62% ((26/42, 95% confidence interval: 47.2-76.6%), and the median survival time was 444days. Our phase I/II trial showed S-1 combined with CPT-11 is effective for AGC and well tolerated with acceptable toxicity. We also evaluated gene expressions of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), thymidine phosphorylase (TP), topoisomerase-I (Topo-I) in patients with gastric cancer treated by this chemotherapy retrospectively. TS mRNA of responding tumors was lower than that of non-responding ones. However, there was no statistically significant difference in overall survival time and time to progression, compared high TS group with low one.