Influenza A virus replication and packaging is mediated by cis-acting signals, which are located at the 3' and the 5' end of the viral segments. The terminal residues can be divided into conserved and nonconserved residues. We have constructed a mutant influenza A/WSN/33 virus, which contains multiple mutations in the nonconserved residues of the neuraminidase (NA) segment. This virus shows a segment-specific reduction of the genomic RNA content in the infected cell and in the progeny virus. Further mutants and revertant viruses revealed that it was not possible to define specific residues, which were responsible for the reduction of the NA-specific RNA. Thus, it appears that an efficient vRNA formation is dependent on the synergistic effect of the terminal sequences.