In vertebrate cells, the nuclear entry of Cdc2-cyclin B1 (MPF1-3) during prophase(4-6) is thought to be essential for the induction and coordination of M-phase events(7-12). Phosphorylation of cyclin B1 is central to its nuclear translocation(8,13,14), but the kinases that are responsible remain unknown. Here we have purified a protein kinase from Xenopus M-phase extracts that phosphorylates a crucial serine residue (S147) in the middle of the nuclear export signal sequence(10,13,15) of cyclin B1. We have identified this kinase as Plx1 (ref. 16), a Xenopus homologue of Polo-like kinase (Plk)-1 (refs 17, 18). During cell-cycle progression in HeLa cells, a change in the kinase activity of endogenous Plk1 toward S147 and/or S133 correlates with a kinase activity in the cell extracts. An anti-Plk1 antibody depletes the M-phase extracts of the kinase activity toward S147 and/or S133. An anti-phospho-S147 antibody reacts specifically with cyclin B1 only during G2/M phase. A mutant cyclin B1 in which S133 and S147 are replaced by alanines remains in the cytoplasm, whereas wild-type cyclin B1 accumulates in the nucleus during prophase. Co-expression of constitutively active Plk1 stimulates nuclear entry of cyclin B1. Our results indicate that Plk1 may be involved in targeting MPF to the nucleus during prophase.