LIM-kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of EGFP-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2, but not LIMK1. One site at Ser283 is present between the PDZ and the kinase domain, and the other site at Thr494 is within the kinase domain. Activation of PKC by phorbol ester- treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser283, and inhibited nuclear import of LIMK2 and the PDZ-kinase construct of LIMK2 (aa 142-638) but not of LIMK1. The PKC- isoform phosphorylated LIMK2 at Ser283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser283 but not Thr494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C dependent phosphorylation of Ser283. Our study shows that phorbol ester or serum stimulation of endothelial cells inhibits nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-mediated phosphorylation of Ser283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1 to S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.