Abstract: The Saccharomyces cerevisiae Start repressor Whi5, the functional analogue of mammalian pRB, shuttles between the nucleus and the cytoplasm throughout the cell cycle: enters into the nucleus at the end of mitosis and remains nuclear until Start. We studied the mechanisms involved in this spatial regulation. The nuclear import depends on the beta-karyopherins of the classical import pathway Kap95 and Cse1. Whi5 contains a monopartite and a bipartite classical NLS localized in its N-terminal region which are functionally redundant. A fragment of Whi5 containing these NLSs is able to constitutively accumulate a GFP(4) protein inside the nucleus throughout the cell cycle, which suggests that the Whi5 nuclear import is not cell cycle-regulated. The nuclear export of Whi5 is assisted by beta-karyopherin Msn5. A two-hybrid assay indicates a physical interaction between Whi5 and Msn5. We identified a fragment of Whi5 with export activity from amino acids 51 to 167. Interestingly, this region drives the export of a chimeric nuclear protein in a cell cycle-regulated pattern similarly to that observed for Whi5. Moreover, the nuclear export driven by Whi5(51-167) depends on the phosphorylation of specific Ser residues. Finally, we identified Cdc14 as the phosphatase required for the nuclear accumulation of Whi5.