The genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. The signals required for replication, transcription, and packaging of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form "panhandle" or "fork" structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. In contrast, the nonconserved noncoding nucleotides located close to the open reading frame of the viral RNAs had not been implicated in RNA transcription. Using a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these nonconserved noncoding regions. Deletion either of the nucleotide residues between the poly(U) stretch and the stop codon at the 5' end or of the nucleotides between position 15 and the start codon at the 3' end did not affect the amount of NA-RNA species found in virions or infected cells. However, a combination of deletions at both the 3' and the 5' ends decreased by 60 times the levels of NA-specific viral RNA found in infected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady-state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of infuenza A/WSN/33 virus also showed a reduction in the NA-specific viral RNA in virions and in infected cells. The present results demonstrate that the nonconserved nucleotides at the 3' and 5' ends of the NA-RNA segment of influenza A virus play an important role in the replication of this segment.