Objectives: To evaluate the response of human cholangoicarcinoma cells to TMX treatment through the Fas pathway by pretreatment with IFN-γ. Summary Background Data: Cholangiocarcinoma remains one of the most difficult tumors to treat in clinical medicine. Currently, there are no effective chemotherapy treatments for this disease. Surgery offers the only opportunity for a cure, with the majority of patients failing to qualify for such treatment. This study seeks to evaluate a potential new modality for treatment of this disease. Methods: Human cholangiocarcinoma cells were treated with anti Fas mab and sorted to two populations (Fas-positive and Fas-negative) by FAC analysis. In vitro individual cell populations were pretreated with IFN-γ 250 units/mL x 18hs. The treated cells assayed for caspase 3, 7, 8, Bak, and for apoptosis with Annexin V after treatment with or without TMX. In Vivo 2 x 106 5 SK-ChA-1 Fas-negative cells were injected into nude mice for development of tumor xenografts. Mice received either no treatment or intra tumor IFN-γ and/or intra peritoneal TMX. Results: More than 90% (90% ± 3.5%) of Fas-positive and 70% (71 ± 2.3%) of Fas-negative cells underwent apoptosis after TMX treatment when pretreated with IFN-γ. In contrast, TMX alone and IFN-γ alone stimulated apoptosis by only 22% (22 ± 3%) P < .00013, and 17% (17 ± 2%) P < .0001 in Fas-ve cells respectively. In vivo human cholangiocarcinomas xenograft growth was significantly inhibited by a combination of TMX + IFN-γ compared to controls P < .0007. Conclusion: TMX exposure to human cholangiocarcinoma after pretreatment with IFN-γ allows for induction of apoptosis in vitro and significant inhibition tumor xenograft growth. The combination of these two compounds may provide novel treatment regimen for cholangiocarcinoma.