The prognosis of patients with biliary tract adenocarcinomas (BTA) is still poor due to lack of effective systemic treatment options. Knowledge of the molecular mechanisms involved in the pathogenesis of this disease is of importance for the development of new treatment strategies. We determined the expression of epidermal growth factor receptor (EGFR) and activated mammalian target of rapamycin (p-mTOR) in paraffin-embedded surgical specimens of BTA (n = 89) by immunohistochemistry. Overall survival was analyzed with Cox models adjusted for clinical and pathologic factors. Combined EGFR/p-mTOR expression was significantly associated with relapse-free survival [adjusted hazard ratio for relapse, 2.20; 95% confidence interval (95% CI), 1.45-3.33; P < 0.001] and overall survival (adjusted hazard ratio for death, 2.32; 95% CI, 1.50-3.58; P < 0.001) of the patients. The effect of the EGFR inhibitors erlotinib or cetuximab and the mTOR inhibitor rapamycin on growth and survival of five BTA cell lines was tested in short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and long-term colony formation assays. Simultaneous blockade of EGFR and mTOR in biliary tract cancer cell lines results in a synergistic inhibition of both phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways, leading to reduced cell growth and survival. These results suggest that combined targeted therapy with EGFR and mTOR inhibitors may potentially benefit patients with BTAs and should be further evaluated in clinical trials.