In human parainfluenza virus type 2 (hPIV-2)-infected cells, anti-phosphoprotein (P)-specific monoclonal antibody (MAb) densely stained the perinuclear regions of infected cells throughout infection, indicating that the P protein was localized exclusively in the cell cytoplasm. By contrast, antigens recognized by MAbs directed against the P-V-common domain of hPIV-2 were located predominantly in the cytoplasm, but in some hPIV-2-infected cells they were also found in the nuclei, suggesting that a fraction of hPIV-2V protein is localized there. hPIV-2 V protein expressed from a cDNA clone was localized in the nuclei of transfected cells. By using indirect immunofluorescence analyses, we examined the intracellular localization of various sequentially deleted V proteins, to determine the nuclear localization signals (NLS) of the V protein. Two noncontiguous regions in the V protein were required for nuclear localization and retention, since deletion of these regions [region I (aa 1–46) and region II (aa 175–196)] resulted in cytoplasmic localization. Both regions resulted in nuclear localization independently. A nucleoplasmin-like NLS was identified in region II but no consensus targeting sequence could be found in region I. When NP protein was co-expressed with V protein or the N-terminal fragment (aa 1–46) of V protein, a fraction of the NP protein was translocated into cell nuclei.