The principles of influenza A virus replication and packaging are not fully understood. In order to investigate the signals required for these processes we have introduced mutations in the terminal non-coding region of an influenza A virus neuraminidase (NA) gene. Specifically, we have obtained two viruses, NA/X and NA/Y, which produced a reduced amount of NA-specific genomic RNA in infected cells but not in the viral particle. These data indicate that (i) specific signals which affect the amount of RNA in the viral particle are distinct from those required for viral replication and (ii) the amount of packaged RNA is not strictly dependent on the amount of RNA produced during replication. In addition, mutant NA/Y was shown to be effectively attenuated in mice. Thus, diminished replication of one viral segment might be a principle on which to base a live influenza virus vaccine.