Influenza A virus non-structural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin alpha isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin alpha/beta pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine/lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35-41, which also function in the dsRNA-binding activity of the NS1A protein. NLS2 was created by a seven amino acid long C-terminal extension (residues 231-237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231 and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal (NoLS), a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionally well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.