UNLABELLED: Recent outbreaks of avian influenza are being caused by unusually virulent H5N1 strains. It is unknown what makes these recent H5N1 strains more aggressive than previously circulating strains. Here, we have compared more than 3000 RNA sequences of segment 8 of type A influenza viruses and found a unique single nucleotide substitution typically associated with recent H5N1 strains. By phylogenetic analysis, biochemical and biophysical experiments, we demonstrate that this substitution dramatically affects the equilibrium between a hairpin and a pseudoknot conformation near the 3' splice-site of the NS gene. This conformational shift may have consequences for splicing regulation of segment 8 mRNA. Our data suggest that besides changes at the protein level, changes in RNA secondary structure should be seriously considered when attempting to explain influenza virus evolution. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.