The human protein kinase Plk1, a member of the polo-like kinase family, is known to function at mitosis. Here we show that the relative specific activity of Plk1 increases in mitosis, that Plk1 is specifically phosphorylated during mitosis, and that phosphatase treatment reduces mitotic Plk1 activity to interphase levels. To identify domains involved in the regulation of Plk1 activity, deletion mutants of Plk1 were constructed and their activities examined. Deletion of the extreme C-terminus of Plk1 substantially increased kinase activity, indicating that the C-terminus harbors an inhibitory domain. Finally, the consequences of over-production of wild-type and mutant Plk1 protein were analyzed, using transient transfection assays. Cells overexpressing Plk1 protein were able to enter mitosis and establish an apparently normal bipolar spindle. In contrast, progression through mitosis was transiently delayed, and cytokinesis appeared to be disturbed, as reflected by a significant increase in large cells with multiple, often fragmented nuclei. These results are relevant to recently proposed roles for Plks during both entry into and exit from mitosis.