The NS2 protein, one of the two nonstructural proteins of influenza A virus, was found to exist in virus particles. By immunochemical method, the average number of NS2 in a virus particle was estimated to be 130-200 molecules. After solubilization of viral envelope, NS2 was still associated with ribonucleoprotein cores, but was later dissociated upon removal of the M1 protein. A filter-binding assay in vitro indicated direct protein-protein contact between M1 and NS2. Following chemical cleavage of the M1 protein, NS2 bound only a C-terminal fragment of M1. By immunoprecipitation, NS2-M1 complexes were also detected in virus-infected cell lysates. These observations altogether indicate specific molecular association between M1 and NS2. Biological significance of M1-NS2 interaction is discussed.