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The expression levels of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil-based chemotherapy in patients with cancer. We herein investigated the differences in the mRNA levels of these enzymes in non-small-cell lung cancer (NSCLC) and evaluated their prognostic value for NSCLC treated by surgical resection. The intratumoral mRNA levels of TP, DPD, TS, and OPRT were quantified in 66 patients with pathological stage I and II NSCLC (adenocarcinoma or squamous cell carcinoma) following complete resection according to the Danenberg Tumor Profile method. The TP level was the only significant prognostic factor for disease-specific survival (DSS) following complete resection; the mean TP mRNA level differed significantly between the high and low mRNA expression groups. The DSS at 5 years was significantly higher in the low TP mRNA compared with that in the high TP mRNA expression group (83.4 vs. 58.6%, respectively; P=0.005). A Cox proportional hazards model revealed that pathological stage, sex, and TP expression were independent prognostic factors for DSS in patients with stage I and II NSCLC following complete resection. Thus, TP level may be used to monitor treatment efficacy and predict the outcome of NSCLC patients.