This report describes the successful generation of an influenza B transfectant virus altered in RNA segment 6, which encodes the neuraminidase (NA) protein. The procedure for selection of the transfectant virus relies on the use of strain-specific anti-NA monoclonal antibodies to inhibit growth of the helper virus within the system. A transfectant virus has been engineered which has a coding change in the NA protein. This change resulted in attenuated growth in vitro that could be rescued by addition of exogenous bacterial NA. The mutant virus-associated NA activity was unstable as a result of the engineered changes. The ability to genetically manipulate influenza B virus segment 6 will allow us to assess the function of both NA and the small protein NB, also coded from this RNA, within the context of the virus infectious cycle.