5-fluorouracil (5-FU) has been used widely, including in gastrointestinal cancer, breast cancer, and non-small cell lung cancer (NSCLC), and its efficacy has been reported to be associated with intratumoral expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). In order to clarify the clinical value of their expression in NSCLC patients treated with 5-FU, we investigated intratumoral expression of TS and DPD immunohistochemically. Of the 68 tumors studied, 40 carcinomas (58. 8%) had high TS expression, and 33 carcinomas (48.5%) had high DPD expression. The 5-year survival rate of the patients with high-TS tumors was significantly lower than that of the patients with low-TS tumors (P=0.0267), and the 5-year survival rate of the patients with high-DPD tumors was significantly lower than that of the patients with low-DPD tumors (P=0.0268). The Cox regression analysis of prognostic variables for NSCLC patients treated with 5-FU demonstrated that the simultaneous evaluation for both TS and DPD expression was found to be a significant indicator of a poor prognosis (P=0.0043). Our results demonstrated that the evaluation of intratumoral TS and DPD activity can be used to accurately predict responsiveness to 5-FU-based chemotherapy in NSCLC patients.