The immediate-early protein (IE180) of pseudorabies virus (PrV) is localized predominantly in the nuclei of infected cells. To define the nuclear localization signals within IE180, we prepared truncated mutants of IE180 and analyzed their localization in the transfected cells by indirect immunofluorescence. Analysis of mutants truncated from the carboxy-terminal end of the 1460-amino acid polypeptide showed that two regions including a short sequence of basic amino acid residues were associated with the nuclear localization of IE180. To assess whether these regions substantially function as signals for nuclear localization of the IE180 molecule, we then constructed two deletion mutants lacking each region. A mutant lacking amino acids 333 to 575 was detected in the nuclei of the transfected cells, whereas the other mutant lacking amino acids 900 to 950 was detected mainly in the cytoplasm. These results suggest that the region of amino acids 900 to 950 is responsible for nuclear localization of IE180.