We previously demonstrated cytotoxicity to cholangiocarcinoma cells via adenoviral delivery of cytosine deaminase gene (AdCMVCD). The biliary milieu may present obstacles to gene transfer in situ. This study determined whether toxin gene (CD or herpes simplex virus thymidine kinase (HSV-tk)) transfer to cholangiocarcinoma cells occurs in human bile. Human cholangiocarcinoma (SK- ChA-1 and Oz) and HeLa cells were infected in Optimem or 10% human bile (filter sterilized and diluted) with an adenoviral vector, AdCMVLacZ encoding the LacZ gene, then FACS analysis performed. Cholangiocarcinoma cells were infected with AdCMVCD, or AdCMVHSV-tk, in Optimem or 10% bile, and treated with 5-FC or ganciclovir (GCV), then proliferation assays performed. SK-ChA- 1 and Oz cells were efficiently transduced by AdCMVLacZ in the presence of human bile (98% and 78%), compared to Optimem (98% and 85%). For SK-ChA-1 cells infected with AdCMVCD and exposed to 5-FC, 84% cytotoxicity occurred in bile compared to 62% in Optimem (P<0.0001). For Oz cells infected with AdCMVHSV-tk and exposed to GCV, 40% cytotoxicity occurred in bile compared to 64% in Optimem (P < 0.0001) but was not synergistic. These results demonstrate effective toxin gene function in a biliary physiologic context.