5-Fluorouracil (5-FU) has been used as a chemotherapeutic drug for colorectal cancer. Escherichia coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, converts 5-FU into 5-fluorouridine monophosphate (5-FUMP) at the initial step of 5-FU activation. We investigated the effects of adenoviral-mediated transfer of the E. coli UPRT gene into human colon cancer cells on 5-FU metabolism and 5-FU chemosensitivity. Three cell lines were used (HT29, KM12 and SW1116). The intracellular levels of 5-fluorodeoxyuridine monophosphate (5-FdUMP) and 5-FU incorporated into RNA after 5-FU treatment in cells infected with adenovirus containing the UPRT gene (AdCA-UPRT) were significantly higher than those of non-infected cells. This was accompanied by marked inhibition of thymidylate synthase (TS) in all cell lines. Furthermore, HT29, KM12 and SW1116 infected with AdCA-UPRT were, respectively, 13.1-, 30.2- and 70.5-fold more sensitive to 5-FU than non-infected cells. Most importantly, treatment with AdCA-UPRT and 5-FU effectively inhibited the growth of HT29-xenografted subcutaneous tumours in nude mice. Therefore, AdCA-UPRT/5-FU treatment had the potential to enhance the actions of 5-FU at both the DNA and RNA levels. Treatment augmented the sensitivity of human colon cancer cells to 5-FU both in vitro and in vivo. We conclude that adenoviral-mediated transfer of the E. coli UPRT gene into colon cancer cells can achieve biochemical modulation of 5-FU and this provides a new approach in the treatment of colorectal cancer.