Abstract Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease with poor prognosis. Treatment with gemcitabine, the FOLFIRINOX scheme or nab-paclitaxel offer only a modest increase in overall survival. For a number of other carcinomas, tumor subtypes have been uncovered that allow the use of targeted therapies. Although subtypes of PDAC were described, this malignancy is clinically still treated as a single disease. We established patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Patients bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. The xenobiotic enzyme, cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Additionally, retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly found in those patient tumors classified as exocrine-like. Whereas the hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. Interfering with these regulatory mechanisms may provide an alternative approach to suppress the CYP3A5 pathway. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. Citation Format: Elisa M. Noll, Christian Eisen, Albrecht Stenzinger, Elisa Espinet, Alexander Muckenhuber, Corinna Klein, Vanessa Vogel, Bernd Klaus, Wiebke Nadler, Christoph Rösli, Christian Lutz, Michael Kulke, Jan Engelhardt, Franziska Zickgraf, Octavio Espinosa, Matthias Schlesner, Xiaoqi Jiang, Annette Kopp-Schneider, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Roland Eils, Nathalia A. Giese, Thilo Hackert, Oliver Strobel, Jens Werner, Markus W. Büchler, Wilko Weichert, Andreas Trumpp, Martin R. Sprick.{Authors}. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B77.