We generated novel recombinant influenza A viruses (vNA38) harboring dicistronic NA segments with an extended native 5' terminal sequence of 70 nucleotides comprised of the last 42 nucleotides of the NA ORF and the 5' noncoding region (5' NCR). vNA38 viruses replicated stably and more efficiently than vNA35 viruses with a dicistronic NA segment comprised of the native 5' NCR only, that we described previously (Vieira Machado, A., Naffakh, N., van der Werf, S., Escriou, N., 2003. Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter. Virology 313, 235-249). In addition, vNA38 viruses drove the expression of higher levels of encoded heterologous proteins than corresponding vNA35 viruses, both in cell culture and in the pulmonary tissue of infected mice. These data demonstrate that a sequence overlapping 5' coding and noncoding regions of the NA segment determines efficient replication and/or propagation of the vRNA. Intranasal immunization of mice with live vNA38 viruses induced B and T cell responses specific for the heterologous protein expressed, establishing the usefulness of such recombinant influenza viruses with a dicistronic segment for the development of live bivalent vaccines.