Crystal structures of ligand-free wild-type and activated zebrafish Plk1 kinase domains reveal the organization of the secondary structural elements around the active site. The cocrystal structure of wild-type Plk1 with ADP documents the hydrolysis of ATP and reveals the autophosphorylation site, while the cocrystal structure of the activated kinase domain with wortmannin, a covalent inhibitor of the PI3 kinase, shows the binding mode of the small molecule to the enzyme and could facilitate the design of more potent Plk1 inhibitors.