Thermosensitive mutations in the DBF2 gene arrest the cell cycle during nuclear division. Although the chromatin has divided in arrested cells, an elongated mitotic spindle is present and Cdc28 protein kinase activity remains high, indicating that nuclear division is incomplete. By execution point analysis we show that Dbf2 carries out an essential cell cycle function after the metaphase to anaphase transition and is therefore required during anaphase and/or telophase. This cell cycle stage-specific requirement for the function of Dbf2 coincides with the cell cycle regulation of Dbf2/Dbf20 protein kinase activity, which can be detected in immunoprecipitates containing Dbf2 or Dbf20. The kinase activity is specific for serine/threonine residues and Dbf2 accounts for the bulk of the activity, with Dbf20 playing a minor role. Furthermore, Dbf2 is a phosphoprotein and, significantly, the dephosphorylated form appears with the same cell cycle timing as the kinase activity, suggesting a role for dephosphorylation in the activation mechanism. In addition, we show that the DBF2 transcript, which is under cell cycle control, is expressed in advance of the activation of the kinase, but that cell cycle-regulated expression of the mRNA is not required for activation of the Dbf2 kinase during M phase. Thus, Dbf2/Dbf20 kinase activity is precisely regulated in the cell cycle by a post-translational mechanism and phosphorylates its target substrates for an event that occurs during anaphase and/or telophase.