5-Fluorouracil (5-FU) has been used worldwide, and the correlation between its effects and thymidylate synthase (TS) expression has been reported in gastrointestinal malignancy. But the significance of TS expression for 5-FU-based chemotherapy has rarely been reported in non-small cell lung cancer (NSCLC). We investigated surgically resected specimens of 23 consecutive patients with previously untreated NSCLC. We used immunohistochemistry and western blot analysis with anti-TS polyclonal antibody to evaluate the existence of TS, and fluorodeoxyuridine-5'-monophosphate (FdUMP) binding assay to evaluate the enzymic activity of TS. We found that 14 samples (60.9%) were positive immunohistochemically, and that the results of immunohistochemistry closely reflected the enzymic activity measured by FdUMP binding assay (ranging from 1.8 to 56.9 pmol/g protein). These results seem to support our experience that 5-FU and its derivatives are clinically significantly effective as a postoperative adjuvant chemotherapy against NSCLC.