The cyclin-dependent kinase 1 (Cdc2)/cyclin B1 complex performs cardinal roles for eukaryotic mitotic progression. Phosphorylation of four serine residues within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at the G(2)/M transition. Still, the role of individual phosphorylation sites and their corresponding kinases remain to be elucidated. Polo-like kinase 1 (Plk1) shows a spatial and temporal distribution which makes it a candidate kinase for the phosphorylation of cyclin B1. We could demonstrate the interaction of both proteins in mammalian cells. Plk1 phosphorylated wild-type cyclin B1 expressed in bacteria and in mammalian cells. Ser-133 within the cytoplasmic retention signal (CRS) of cyclin B1, which regulates the nuclear entry of the heterodimeric complex during prophase, is a target of Plk1. In contrast, MAPK (Erk2) and MPF phosphorylate Ser-126 and Ser-128 within the CRS. Phosphorylation of CRS by MAPK (Erk2) prior to Plk1 treatment induced enhanced phosphorylation of cyclin B1 by Plk 1 suggesting a synergistic action of both enzymes towards cyclin B1. In addition, pretreatment of cyclin B1 by MAPK (Erk2) altered the phosphorylation pattern of Plk 1. Mutation of Ser-133 to Ala decreased the phosphorylation of cyclin B1 in vivo. An immunofluorescence study revealed that a mutation of Ser-133 reduced the nuclear import rate of cyclin B1. Still, multiple serine mutations are required to prevent nuclear translocation completely indicating that orchestrated phosphorylation within the CRS triggers rapid import of cyclin B1.