Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.