The formation of influenza virus ribonucleoprotein (RNP) is a necessary step in viral assembly and maturation in infected cells, but the mechanism remains incompletely understood. Influenza virus proteins such as matrix (M1) and cellular proteins have been implicated in assembly and transport of RNP. To study the assembly of RNP and the translocation of RNP complexes in cells, RNPs were reconstituted from nucleoprotein (NP), M1, and viral RNA (vRNA) synthesized in vitro. The syntheses were accomplished using specific plasmids in a system coupling transcription and translation under the control of the T7 promoter. The density of the resulting RNP complexes was analyzed by glycerol gradient centrifugation and the morphology was examined by transmission electron microscopy. Protomers of NP self-assembled into circular oligomers regardless of the presence of vRNA or M1. However, helical structures similar in conformation and density to RNPs purified directly from influenza virus were formed only when M1 and vRNA were also present. In the absence of vRNA, no helical structures were formed from NP and M1. The plasmids also contained the CMV promoter, which permitted expression of M1, NP, and vRNA in Madin-Darby canine kidney (MDCK). M1 and NP were both present in the cytoplasm of MDCK also expressing vRNA, but NP was retained in the nucleus of cells expressing M1 without vRNA. Our data demonstrate for the first time that vRNA and M1 together promote the self-assembly of influenza virus NP into the quaternary helical structure typical of the viral RNP. The results also indicate that the interaction of NP with vRNA and M1 in a system devoid of other viral proteins can lead to translocation of RNP from nucleus to cytoplasm.