Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48-NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.