The Borna disease virus (BDV) replicates in the nucleus. The viral p40 protein (N), which is found abundantly in the nucleus in BDV-infected cells, may play an important role in virus replication. To analyze the amino acid residues involved in the nuclear targeting of BDV N, a series of eukaryotic expression plasmids encoding deletion mutants of N was constructed and transfected into COS-7 cells. In indirect immunofluorescence assays with a rabbit anti-BDV N antiserum, wild-type N was located in the nucleus of transfected cells in the absence of other viral constituents. In contrast, mutants lacking the 13 NH2-terminal amino acid residues 1MPPKRRLVDDADA13 in common gave a cytoplasmic localization pattern. Similarly, a mutant with substitution of 4KRR6 by 4NSG6 was retained in the cytoplasm. Furthermore, a nonapeptide, 3PKRRLVDDA11, derived from the NH2-terminal region of N conferred nuclear targeting activity to beta-galactosidase, which normally resides in the cytoplasm. Thus, we have identified the nuclear targeting signal of the BDV N and narrowed it to the NH2-terminal region where 4KRR6 basic amino acid residues are located.