The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.