The development of new antiviral molecules to fight the possible emergence of influenza viruses with pandemic potential is needed. In this study, phosphorothioate oligonucleotides (S-ONs) derived from the packaging signals in the 3' and 5' ends of the viral PB2 RNA were associated with liposomes and tested against influenza virus in vitro. A 15-mer S-ON derived from the 5' end of the viral PB2 RNA, complementary to the 3' end of its coding region (nucleotides 2279-2293) and designated 5-15b, proved markedly inhibitory. The antiviral activity of 5-15b was dose- and time-dependent but was independent of the cell substrate and multiplicity of infection used. Importantly, inhibition of influenza A and B viruses required S-ONs reproducing the respective packaging signals. Furthermore, 5-15b and its antisense derivative S-ON activity did not affect intracellular accumulation of viral RNA. Confocal microscopy showed that 5-15b is clearly nucleophilic. These findings indicate that the packaging signal at the 5' end of the PB2 RNA is an interesting target for the design of new anti-influenza-virus compounds.