Cholangiocarcinoma is a malignancy that is resistant to current therapy. We applied the toxin gene therapy strategy of cytosine deaminase conversion of the nontoxic prodrug 5-fluorocytosine to 5-fluorouracil combined with radiotherapy to cholangiocarcinoma. The transduction efficiency of SK-ChA-1 cholangiocarcinoma cells was determined by fluorescence-activated cell-sorting analysis following infection with recombinant adenovirus AdCMVLacZ, which encodes the gene for β-galactosidase. To evaluate cytosine deaminase-mediated conversion of 5-fluorocytosine to 5-fluorouracil and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes cytosine deaminase, and exposed to 5-fluorocytosine for 6 to 8 days. Additive cytotoxicity of radiation therapy was evaluated by cobalt-60 exposure following AdCMVCD infection and 5-fluorocytosine treatment. SK-ChA-1 cells were transduced (98.4%) by AdCMVLacZ at 100 plaque-forming units per cell. Following infection with AdCMVCD and exposure to 5 to 100 μg/ml of 5-fluorocytosine, 20% to 64% of SK-ChA-1 cells were killed. A combination of radiation and cytosine deaminase/5-fluorocytosine therapy resulted in enhanced cell killing (83.5% to 91.5%). Cholangiocarcinoma cells were transduced by recombinant adenoviral vectors and were killed by cytosine deaminase-mediated production of 5-fluorouracil. Enhanced cytotoxicity was seen with the addition of external beam radiation. These results provide a foundation for multimodality therapy for human cholangiocarcinoma that combines gene therapy technology with radiation therapy.