To provide the basis for improved therapeutic benefit in combination chemotherapy with interferon (IFN) and 5-fluorouracil (5-FU), we investigated the modulatory actions of human recombinant IFN alfa-2a on 5-FU in five renal cell carcinoma (RCC) cell lines in vitro, in particular focusing on thymidine phosphorylase (TP) expression.The sensitivity of RCC cell lines to the drugs was evaluated using an AlamarBlue assay. An enzyme-linked immunosorbent assay (ELISA) was used to determine TP expression.IFN-alpha enhanced the sensitivity of three of five RCC cell lines to 5-FU in a dose- and schedule-dependent manner. When IFN-alpha was given prior to 5-FU, sensitivity to 5-FU was significantly higher than when IFN-alpha was given simultaneously (P < 0.05). IFN-alpha enhanced TP expression in a dose-dependent manner in three of five RCC cell lines (P < 0.05). The relative IFN-alpha-induced increase in sensitivity to 5-FU correlated with the relative IFN-alpha-induced increase in TP expression (P < 0.05). In addition, two of three RCC cell lines with more than a twofold increase in sensitivity to 5-FU induced by IFN-alpha showed a higher TP expression without IFN-alpha stimulation.These results suggest that IFN-alpha upregulates TP expression and modulates 5-FU anabolism thus enhancing 5-FU cytotoxicity in a dose- and schedule-dependent manner in some RCC cells. The results imply that TP expression measurement in RCC could identify subgroups of metastatic RCC that may respond to IFN-alpha/5-FU combination therapy, and sequential administration of IFN-alpha followed by 5-FU may be beneficial in such cases.