The difference between neurovirulent WSN and nonneurovirulent Hong Kong (HK) virus strains in intracerebral infection of mice was attributed to NA, M, and NS genes ( Sugiura and Ueda, 1980, Virology 101, 440–449). The mechanism by which NA, M, and NS proteins were involved in the virulence was studied by infection of mouse neuroblastoma cells in culture. Replication of prototype and recombinant viruses in neuroblastoma cells reflected in many respects the infection of the mouse brain in situ. Viruses containing WSN neuraminidase multiplied productively in the neuroblastoma cells. Progeny viruses released from neuroblastoma cells infected with viruses containing HK neuraminidase had uncleaved HA polypeptide, and were, as a result, noninfectious. Treatment with trypsin in vitro conferred the infectivity to the noninfectious progeny. The role of WSN neuraminidase was considered to facilitate the cleavage of HA polypeptide. Indeed, the brains of mice infected intracerebrally with HK virus on the previous day contained noninfectious viruses which were activated by trypsin. When either M or NS gene was derived from HK virus, the growth rate of recombinants was decreased in multicycle infection. These recombinants infected neuroblastoma cells with similar efficiency as virulent viruses. M and NS proteins from HK virus probably affected the replicative process in neuroblastoma cells at the stage later than the initiation of infection, resulting in diminished growth potential of the virus.