Because influenza virus replicates in the nucleus and buds from the plasma membrane, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear membrane. Export from the nucleus to the cytoplasm was found to depend on the viral matrix protein (M1). M1 associated with newly assembled viral RNPs (vRNPs) in the nucleus and escorted them to the cytoplasm through the nuclear pores. In contrast, during entry of the virus into a new host cell, M1 protein dissociated from the RNPs, allowing them to enter the nucleus. Amantadine, an antiviral agent that induces an early block in influenza A infection, was found to block the dissociation event and thereby to prevent import of incoming RNPs into the nucleus. Together, these results showed that M1 modulates the directionality of vRNP transport into and out of the nucleus.