Cytochrome P450 2C9 (CYP2C9) metabolizes many clinically important drugs including warfarin and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, found in a warfarin hypersensitive patient with Arg125Leu and Arg144Cys mutations. Here, we have investigated the molecular basis for the functional consequences of these polymorphic changes. CYP2C9.1 and CYP2C9-Arg144Cys expressed in human embryonic kidney 293 cells effectively metabolized both S-warfarin and diclofenac in NADPH-dependent reactions, whereas CYP2C9-Arg125Leu or CYP2C9.35 were catalytically silent. However, when NADPH was replaced by a direct electron donor to CYPs, cumene hydroperoxide, hereby bypassing the CYP oxidoreductase (POR), all variant enzymes were active, indicating unproductive interactions between CYP2C9.35 and POR. In silico analysis revealed a decrease of the electrostatic potential of CYP2C9-Arg125Leu-POR interacting surface and the loss of stabilizing salt bridges between these proteins. In conclusion, our data strongly suggest that the Arg125Leu mutation in CYP2C9.35 prevents CYP2C9-POR interactions resulting in the absence of NADPH-dependent CYP2C9-catalyzed activity in vivo, thus influencing the warfarin sensitivity in the carriers of this allele.The Pharmacogenomics Journal advance online publication, 10 December 2013; doi:10.1038/tpj.2013.41.