We semiquantificated the mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase(OPRT) from the frozen tissue of colorectal cancer, in order to examine the relationships among these enzymes and the efficacy for 5-fluorouracil (FU)-based treatment in terms of the combinations of the expression levels of these enzymes. There were weak and positive relationships among TS mRNA expression and OPRT mRNA expression or DPD mRNA expression, while there was no significant relationship between DPD mRNA expression and OPRT mRNA expression (n = 112). In 39 patients who received 5-FU-based chemotherapy with evaluable lesions, patients with low TS expression (n = 23) showed a higher response rate (52%) as compared to those with high TS expression (13%, p = 0.02). Patients with low TS, low DPD, or high DPD expression tended to show a higher response rate (50%) than those with the other combinations (26%, p = 0.09). In addition, all of the responders with only one favorable factor (n = 4) were associated with low TS or low DPD expression. In conclusion, low TS expression followed by low DPD expression is important to predict the efficacy of 5-FU-based treatment for colorectal cancer. However, it may be of little significance to semiquantify the expression of OPRT.