Influenza virus nucleoprotein (NP), synthesized in Xenopus oocytes after injection of cloned NP cDNA, enters and accumulates in the nucleus. We have used in vitro mutagenesis of this cDNA to study the cellular distribution of mutated NP polypeptides. Mutants lacking amino acids 327-345 of wild-type NP enter the nucleus but do not accumulate there to the same extent as the wild-type protein, suggesting that this region has a role in nuclear accumulation. This possibility is further strengthened by similar studies involving the production of fusion proteins in which various amino-terminal sequences of the NP gene are fused to the complete chimpanzee alpha 1-globin sequence: when globin cDNA was injected into and expressed in oocytes the protein remains exclusively in the cytosol; however, when the globin cDNA is fused to a portion of NP cDNA that includes the region encoding amino acids 327-345, the resulting fusion protein enters and accumulates in the nucleus. Fusion proteins lacking this region of the NP enter but do not accumulate in the nucleus.