The redox potentials of FAD and FMN of Cytochrome P450 reductase (reductase) are equivalent in solution but differ by 138 mV when bound to reductase. The interaction of each flavin with its flavin binding domain confers the unique electron transferring abilities to each flavin. In order to determine flavin binding properties and activity of the FAD binding domain, we have expressed in pTrcHis three fragments (1161, 1244, and 1556 bp) of rat liver reductase cDNA encompassing the proposed FAD and NADPH binding domain. The FAD binding fragments from cells harboring the 1161- and 1556-bp-containing vectors were stable and bound 0.66 and 0.71 mol FAD/mol enzyme, respectively. Both fragments reduce ferricyanide (54 and 104% of FMN-less reductase/mol bound flavin, respectively) and participate in the transhydrogenation reaction of 3-AcPy-ADP (41 and 65% of FMN-less reductase/mol bound flavin, respectively). FAD-less fragments were purified and reconstituted with 8-amino-FAD and 8-chloro-FAD to determine binding efficiencies.