We previously demonstrated that epidermal growth factor (EGF) induces a decrease in dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), in EGF receptor (EGFR)-positive human SKG-IIIb uterine cervical carcinoma cells, and thereby increased the sensitivity of the cells to 5-FU. In the present study, we examined the individual and combined effects of 5-FU and EGF on growth and DPD activity in SKG-IIIb cells, and also investigated the mode of regulation of DPD activity. The cells showed sensitivity to 5-FU, and growth was stimulated by EGF. When the agents were used in combination, the sensitivity of SKG-IIIb cells to 5-FU was increased roughly sixfold at maximum, as judged in terms of the 50% growth-inhibitory concentration. We then examined the effects of 5-FU and EGF on DPD. Either agent inhibited DPD activity and protein expression in a concentration-dependent manner. Expression of DPD mRNA was concentration-dependently inhibited by treatment with 5-FU and by EGF at a concentration that strongly stimulated cell growth. Further, combination treatment inhibited DPD activity, as well as DPD protein and mRNA expression, more strongly than did treatment with 5-FU or EGF alone. These results suggest that inhibition of DPD activity by EGF or 5-FU is regulated at least at the level of protein expression and that regulation via mRNA is also involved. The above findings indicate that 5-FU and EGF act synergistically in suppressing DPD activity and that the use of 5-FU against tumors in which EGF plays an important role would maximize the potential of 5-FU as an anticancer drug.