The γ134.5 protein of herpes simplex virus type 1 (HSV-1) is required for viral neurovirulence in vivo. In infected cells, this viral protein prevents the shutoff of protein synthesis mediated by double-stranded-RNA-dependent protein kinase PKR. This is accomplished by recruiting protein phosphatase 1 to dephosphorylate the α subunit of translation initiation factor eIF-2 (eIF-2α). Moreover, the γ134.5 protein is implicated in viral egress and interacts with proliferating cell nuclear antigen. In this report, we show that the γ134.5 protein encoded by HSV-1(F) is distributed in the nucleus, nucleolus, and cytoplasm in transfected or superinfected cells. Deletion analysis revealed that the Arg-rich cluster from amino acids 1 to 16 in the γ134.5 protein functions as a nucleolar localization signal. The region from amino acids 208 to 236, containing a bipartite basic amino acid cluster, is able to mediate nuclear localization. R215A and R216A substitutions in the bipartite motif disrupt this activity. Intriguingly, leptomycin B, an inhibitor of nuclear export, blocks the cytoplasmic accumulation of the γ134.5 protein. L134 A and L136 A substitutions in the leucine-rich motif completely excluded the γ134.5 protein from the cytoplasm. These results suggest that the γ134.5 protein continuously shuttles between the nucleus, nucleolus, and cytoplasm, which may be a requirement for the different activities of the γ134.5 protein in virus-infected cells.