The cDNA encoding the murine Mx1 protein, a mediator of resistance to influenza virus, was inserted into a replication-competent avian retroviral vector in either the sense (referred to as Mx+) or the antisense (referred to as Mx-) orientation relative to the viral structural genes. Both vectors produced virus retaining the Mx insert (Mx recombinant viruses referred to as Mx+ and Mx-) following transfection into chicken embryo fibroblasts (CEF). Mx protein of the appropriate size and nuclear localization was expressed only in CEF cells infected with the Mx+ virus. Mx expression was observed in all Mx+-infected cells and was stable during long-term culture. Cells infected with the Mx+ virus were resistant to infection by human influenza A/WSN/33 (H1N1) and avian influenza viruses A/Turkey/Wisconsin/68 (H5N9) and A/Turkey/Massachusetts/65 (H6N2), but were susceptible to infection by the enveloped RNA viruses Sindbis and vesicular stomatitis virus (VSV). Normal CEF and cells infected with the Mx- virus were susceptible to influenza A, Sindbis, and VSV. The synthesis of influenza proteins, especially the larger polymerase and hemagglutinin proteins, was reduced in Mx+ retrovirus-infected cells superinfected by influenza A. © 1991.