Recent clinical studies have indicated that intra-tumoral gene expression levels of 5-fluorouracil (5-FU) metabolism-related enzymes may predict the clinical response of several cancers to 5-FU-based chemotherapy. However, few studies examining oral squamous cell carcinomas (OSCCs) have been reported. In this study, we determined the expression levels of 5-FU metabolism-related enzymes like thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylate (TP) and orotate phosphoribosyl transferase (OPRT) using reverse transcription-polymerase chain reaction (RT-PCR) combined with laser capture microdissection (LCM). We also evaluated the correlation between the mRNA expressions of these genes and clinico-pathological factors or the treatment effects of 5-FU-based chemotherapy combined with radiotherapy in 27 patients with OSCC. No significant correlation was observed between the mRNA expression levels of any of the examined genes and the T-stage, N-stage, differentiation grade or mode of tumor invasion. Although TS and OPRT mRNA were not correlated with the histopathological effects and the development of tumor recurrence, DPD and TP mRNA were significantly correlated with the histopathological effects and tumor recurrence. A significant positive correlation was also observed between the expression of TS and DPD mRNA, but no other correlations were observed among the other genes. Our results suggest that the combined evaluation of TP and DPD mRNA expression in tumor cells using LCM and RT-PCR may be a useful predictor of the efficacy of 5-FU-based chemotherapy combined with radiotherapy in patients with OSCC.