The CDC25B dual specificity phosphatase is involved in the control of the G2/M transition of the cell cycle. Subcellular localization might represent an important aspect of the regulation of its activity. We have examined in transiently transfected asynchronous HeLa cells the localization of HA-tagged CDC25B proteins and found that they are nuclear or cytoplasmic suggesting the existence of an active shuttling. Accordingly, localization analysis of deletion and truncation proteins indicates that CDC25B contains a putative nuclear localization signal located between residues 335 and 354. We also demonstrated that a short 58 residues deletion of the amino-terminus end of CDC25B is sufficient to retain it to the nucleus. Mutational analysis indicates that a nuclear export sequence is located between residues 28 and 40. In addition, treatment of the cells with the exportin inhibitor, Leptomycin B, has the same effect. The mutation of Ser-323, a residue that is essential for the interaction with 14-3-3 proteins, also abolishes cytoplasmic staining. The subcellular localization of CDC25B is therefore dependent on the combined effects of a nuclear localization signal, a nuclear export signal and on the interaction with 14-3-3 proteins.