Radiosensitization of human gastrointestinal tumors by 5-fluorouracil (5-FU) has been studied in vitro and clinically in human cancer therapy trials. The bacterial enzyme cytosine deaminase (CD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) into 5-FU. Human colon cancer cells stably expressing CD have been shown by other investigators to be sensitized to radiation following treatment with 5-FC. We previously used an adenoviral vector under control of the cytomegalovirus promoter (AdCMVCD) encoding the CD gene in combination with 5-FC and a single fraction of radiation exposure to enhance cytotoxicity to human cholangiocarcinoma cells in vitro and in vivo. The purpose of this study was to determine whether AdCMVCD infection and 5-FC with multiple fraction low-dose radiotherapy results in enhanced cytotoxicity. In the present study, we utilized AdCMVCD and 5-FC with single fraction radiotherapy to demonstrate enhanced cytotoxicity to WiDr human colon carcinoma cells in vitro. Additionally, we tested this gene therapy/prodrug treatment strategy employing a fractionated radiation dosing schema in animal models of WiDr colon carcinoma and SK-ChA-1 cholangiocarcinoma. A prolonged WiDr tumor regrowth delay was obtained with AdCMVCD infection in combination with systemic delivery of 5-FC and fractionated external beam radiation therapy compared with control animals treated without radiation, without 5-FC, or without AdCMVCD. The results of treatment with AdCMVCD + 5-FC + radiation therapy to cholangiocarcinoma xenografts were equivalent to those obtained with systemic 5-FU administration + radiation. Thus, the use of AdCMVCD can be effectively combined with clinically relevant 5-FC and radiation administration schemes to achieve enhanced tumor cell killing and increased control of established tumors of human gastrointestinal malignancies.