Chk2 is a nuclear protein kinase involved in the DNA damage-induced ATM-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by GST pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 nuclear localization signals (NLSs), located at a.a. 179-182 (NLS-1), 240-256 (NLS-2) and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in yeast two-hybrid, and in particular with its carboxy-terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with GFP-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.