Recent studies from various eukaryotic model systems indicate that polo-like kinases (Plks) play an ever-increasing role in the regulation of cell cycle progression. Early genetic studies have demonstrated that Cdc5, a budding yeast counterpart of vertebrate Plks, is essential for mitosis. Mammalian Plks primarily localize to the microtubule organization center during interphase and undergo dramatic subcellular relocation during mitotic progression. Many key cell cycle regulators such as p53, Cdc25C, cyclin B, and components of the anaphase promoting complex are directly targeted by Plks. Although the exact mechanisms of action of these protein kinases in vivo remain to be elucidated, Plks appear to orchestrate various cell cycle checkpoints (intra-S phase, G2/M transition, spindle assembly, and cytokinesis checkpoints) that protect cells against genetic instability during cell division.