Polo-like kinase (Plk1) is crucial for cell-cycle progression via mitosis. Members of the Polo-like kinase family are characterized by the presence of a C-terminal domain termed the Polo-box domain (PBD) in addition to the N-terminal kinase domain. The PBD of Plk1 was cloned and overexpressed in Escherichia coli. Crystallization experiments of the protein in complex with an unphosphorylated and a phosphorylated target peptide from Cdc25C yield crystals suitable for X-ray diffraction analysis. Crystals of the PBD in complex with the phosphorylated peptide belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 38.23, b = 67.35, c = 88.25 angstroms, alpha = gamma = beta = 90 degrees, and contain one molecule per asymmetric unit. Crystals of the PBD in complex with the unphosphorylated peptide belong to the monoclinic space group P2(1), with unit-cell parameters a = 40.18, b = 49.17, c = 56.23 angstroms, alpha = gamma = 90, beta = 109.48 degrees, and contain one molecule per asymmetric unit. The crystals diffracted to resolution limits of 2.1 and 2.85 angstroms using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS), respectively.