Polo kinase is activated as cells enter mitosis and plays a central role in coordinating diverse mitotic events, yet the mechanisms leading to activation of Polo kinase are poorly understood . Work in Xenopus meiotic cell cycles has suggested that Polo kinase functions in a pathway that helps trigger activation of Cdk1 . However, studies in other organisms have suggested that activation of Polo kinase is dependent upon Cdk1 and therefore occurs downstream of Cdk1 activation . In this study, we have investigated the role of Cdk1 in the activation of budding yeast Polo kinase. The budding yeast homologs of Cdk1 and Polo kinase are referred to as Cdc28 and Cdc5. We show that signaling from Cdc28 is required to maintain Cdc5 activity in vivo. Furthermore, purified Cdc28 associated with the mitotic cyclin Clb2 is sufficient to activate purified Cdc5 in vitro. A single Cdc28 consensus phosphorylation site found at threonine 242 in the activation loop segment of Cdc5 is required for Cdc5 function in vivo and for kinase activity in vitro, whereas four other Cdc28 consensus sites are dispensable. Analysis of Cdc5 phosphorylation by mass spectrometry indicates that threonine 242 is phosphorylated in vivo. These results suggest that Cdc28 activates Cdc5 via phosphorylation of threonine 242.