The purpose of this study was to construct an H9N2 virus-based bivalent live vaccine expressing the protective antigen of a different subtype of influenza virus. Reverse genetics was used to generate an influenza virus containing nine gene segments derived from the A/Chicken/Jiangsu/11/2002 (H9N2) strain, including independent M1 and M2 matrix gene segments. A recombinant virus expressing the H1N1 HA1 hemagglutinin protein was produced on this framework by substituting the extracellular domain of the H9N2 M2 gene with the H1N1 HA1 fragment from A/PR/8/34 (PR8, H1N1). The resulting hybrid virus H9N2-PR8/HA1 was genetically stable and of low pathogenicity. Intra-nasal immunization of BALB/c mice with H9N2-PR8/HA1 virus induced both anti-H9N2 virus and anti-PR8 HA antibodies and conferred protection to mice against lethal challenge (40x LD(50)) with either H1N1 or H9N2 viruses. This study provides a new influenza H9N2 virus model for the expression and/or delivery of foreign antigens.