The sensitivity to 5-fluorouracil (5-FU) has been reported to be associated with target molecule thymidylate synthase (TS), fluoropyrimidine-metabolising enzymes such as orotate phosphoribosyltransferase (OPRT), and dihydropyrimidine dehydrogenase (DPD). We performed an immunohistochemical study on the clinical significance of TS, OPRT, and DPD expression using 151 resected non-small-cell lung cancer (NSCLC) patients postoperatively treated with a combination of tegafur and uracil (UFT). Eighty-two carcinomas were TS-positive, 105 carcinomas were OPRT-positive, 68 carcinomas were DPD-positive. No correlation was observed in the HSCORE between the TS and OPRT expression (r=0.203), between the TS and DPD expression (r=0.098), or between the OPRT and DPD expression (r=0.074). Regarding the survival of NSCLC patients treated with UFT, the 5-year survival rate of patients with TS-negative tumours was significantly higher than that with TS-positive tumours (P=0.0133). The 5-year survival rate of patients with OPRT-positive stage II to III tumours was significantly higher than that with OPRT-negative stage II to III tumours (P=0.0145). In addition, the 5-year survival rate of patients with DPD-negative tumours was also significantly higher than that with DPD-positive tumours (P=0.0004). A Cox multivariate regression analysis revealed the TS status (hazard ratio 2.663; P=0.0003), OPRT status (hazard ratio 2.543; P=0.0005), and DPD status (hazard ratio 2.840; P<0.0001) to all be significant prognostic factors for the survival of resected NSCLC patients postoperatively treated with UFT.