5-fluorouracil (5-FU) is a basic agent used in chemotherapy. The aim of this study is to investigate the gene expression of 5-FU anabolic and catabolic enzymes in hepatocellular carcinoma (HCC) and non-tumor tissue, respectively to increase our knowledge of resistant mechanisms to 5-FU in HCC. The relative mRNA level of orotate phosphoribosyltransferase (OPRT), ribonucleotide reductase (RNR), dihydropyrimidine dehydrogenase (DPD) and target enzyme thymidylate synthase (TS), were analyzed in 30 matched samples of HCC (T) and non-tumor tissue (NT) using quantitative RT-PCR. The expression of OPRT, RNR-M1, RNR-M2 and TS is significantly higher in T compared with in NT (1.3-fold increase, 1.6-fold, 7.1-fold, 1.9-fold, respectively), but that of DPD showed no difference between T and NT. Our results show that HCC should not be treated with 5-FU alone because of its instability in liver.