By microinjecting purified glutathione S-transferase linked to all or parts of herpes simplex virus type 1 US11 protein into either the nucleus or the cytoplasm, we have demonstrated that this nucleolar protein exhibits a new type of localization signal controlling both retention in nucleoli and export to the cytoplasm. Saturated mutagenesis combined with computer modeling allowed us to draw the fine-structure map of this domain, revealing a new proline-rich motif harboring both activities, which are temperature dependent and regulated by phosphorylation. Finally, crossing the nuclear pore complex from the cytoplasm to the nucleus is an energy-dependent process for US11 protein, while getting to nucleoli through the nucleoplasm is energy independent.Nucleoli are mainly the seat of ribosome biogenesis, a highly complex process leading to the production of preribosomal particles, which are then released in the nucleoplasm and exported to the cytoplasm as mature ribosomal subunits (1,29,36,48,50). Recently, new data have suggested that the nucleolus might also play a crucial role in several other key events in cell life. By being the site of transient sequestration and maturation of several factors, the nucleolus might be involved in the control of cell division, aging, and mRNA export (26,40,41). In addition, proteins belonging to very distinct species of viruses also have various functions in the nucleolus. These proteins appear to be involved in replication of viral genomes as well as in transcriptional and posttranscriptional regulation of gene expression (11,12,32,44,52,55). As a consequence of the various functions of the nucleolus, large amounts of many different components are imported into and exported out of it.Whereas a clear picture of the general mechanisms governing nucleocytoplasmic transport of proteins is now emerging, how nucleolus components are delivered to their correct site of action for a coordinate activity remains poorly understood (29,50). Crossing the nuclear pore complex (NPC) requires a signal and is mediated by an energy-dependent process (for a review, see references 39 and 21). Prototypic mono-and bipartite classical nuclear localization signals (cNLS) are those found in simian virus 40 large T antigen and in nucleoplasmin.