We examined 116 stage I-IIIA non-small-cell lung cancer (NSCLC) patients for intra-tumoral expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) using TaqMan reverse transcription polymerase chain reaction (RT-PCR) assay to clarify the correlation between gene expression and the efficacy of 5-fluorouracil (5-FU) in patients with NSCLC. Patients who were administered 5-FU alone after surgery comprised the 5-FU group (n = 30), and those who underwent only surgery comprised the control group (n = 86). When dichotomized at the mean TS and DPD mRNA level, patients with low-DPD tumors who were administered 5-FU had a significantly better prognosis than those who did not receive adjuvant treatment (p = 0.041). In addition, in the 5-FU group, 10 patients with both low-TS and low-DPD tumors have not had any relapse, whereas 8 of the 20 patients with either high-TS or high-DPD tumors developed distant metastasis after surgery. Based on these results, the quantitation of TS and DPD mRNA levels may predict the efficacy of 5-FU after surgery for patient with NSCLC.