Polo-like kinase 1 (Plk1) functions as a key regulator of mitotic events by phosphorylating substrate proteins on centrosomes, kinetochores, the mitotic spindle, and the midbody. Through mechanisms that are incompletely understood, Plk1 is released from and relocalizes to different mitotic structures as cells proceed through mitosis. We used fluorescence recovery after photobleaching to examine the kinetics of this process in more detail. We observed that Plk1 displayed a range of different recovery rates that differ at each mitotic substructure and depend on both the Polo-box domain and a functional kinase domain. Upon mitotic entry, centrosomal Plk1 becomes more dynamic, a process that is directly enhanced by Plk1 kinase activity. In contrast, Plk1 displays little dynamic exchange at the midbody, a process that again is modulated by the kinase activity of Plk1. Our findings suggest that the intrinsic kinase activity of Plk1 triggers its release from early mitotic structures and its relocalization to late mitotic structures. To assess the importance of Plk1 dynamic relocalization, Plk1 was persistently tethered to the centrosome. This resulted in a G(2) delay, followed by a prominent prometaphase arrest, as a consequence of defective spindle formation and activation of the spindle checkpoint. The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression. This dependence on dynamic Plk1 was further observed during the mitotic reentry of cells after a DNA damage G(2) checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on the dynamic behavior of Plk1.