Polo-like kinases (Plks) have been implicated in various aspects of M-phase progression in organisms ranging from yeast to man. In vertebrates, Plks participate in centrosome maturation and spindle assembly, as well as the activation of the Cdk1/cyclin B complex. Moreover, Plks are required for the destruction of mitotic cyclins, indicating that they play an important role in the regulation of the ubiquitin-dependent proteolytic degradation machinery that controls exit from M-phase. Here, we have fused Green Fluorescent Protein (GFP) to the N-terminus of human Plk1, and expressed this chimeric construct in human cells. We found that GFP-Plk1 associates with centrosomes, the equatorial spindle midzone and the postmitotic bridge of dividing cells, confirming and extending previous results obtained with conventional immunofluorescence microscopy. In addition, however, we observed fluorescence emanating from the midbody between dividing cells, and from discrete dots associated with mitotic chromosomes. This latter staining pattern being reminiscent of centromeres, we performed double-labeling experiments with antibodies against the centromeric marker CENP-B, and reexamined the subcellular localization of endogenous Plk1 using different fixation procedures. Our data clearly show that both GFP-tagged Plk1 and endogenous Plk1 associate with the kinetochore/centromere region of human mitotic chromosomes. This novel localization of Plk1 suggests that substrates and/or regulators of Plks may be found among kinetochore-associated proteins with important functions in chromosome segregation and/or spindle checkpoint mechanisms.