Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU) and its activity is closely associated with cellular sensitivity to 5-FU. This study examines the role of DPD in the antiproliferative effects of 5-FU combined with IFN-alpha on hepatocellular carcinoma (HCC) cells in culture and asks whether IFN-alpha could affect DPD expression. The combined action of IFN-alpha and 5-FU on three HCC lines was quantified by a combination index method. Coadministration of IFN-alpha and 5-FU showed synergistic effects against HAK-1A and KYN-2 but antagonistic effects against KYN-3. The cellular expression levels of DPD mRNA and protein were markedly up-regulated in KYN-3 cells by IFN-alpha but were down-regulated in HAK-1A and KYN-2. The expression of thymidylate synthase mRNA and protein was down-regulated by IFN-alpha in all three cell lines. Coadministration of a selective DPD inhibitor, 5-chloro-2,4-dihydroxypyridine (CDHP), enhanced the antiproliferative effect of 5-FU and IFN-alpha on KYN-3 approximately 4-fold. However, the synergistic effects of 5-FU and IFN-alpha on HAK-1A and KYN-2 were not affected by CDHP. The antiproliferative effect of 5-FU could thus be modulated by IFN-alpha, possibly through DPD expression, in HCC cells. Inhibition of DPD activity by CDHP may enhance the efficacy of IFN-alpha and 5-FU combination therapy in patients with HCC showing resistance to this therapy.