. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the ␣ subunit is phosphorylated by p34 cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of ␣. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34 cdc2 phosphorylation. Synthetic peptides corresponding to regions of ␣ that contain each of these residues are phosphorylated by p34 cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of ␣ are observed when any of the four residues are mutated to alanine. When all four residues are mutated to alanine, the fusion protein is no longer phosphorylated by p34 cdc2 at any of the sites that are phosphorylated in mitotic cells. These results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the sites on the ␣ subunit of casein kinase II that are phosphorylated in mitotic cells.Biochemical and genetic studies have demonstrated that the p34 cdc2 protein kinase is an indispensable regulator of events leading to the division of eukaryotic cells (for reviews see Refs. 1-4). To ensure that the division of cells is very precisely regulated, the activity of this protein serine/threonine kinase is exquisitely controlled through its interactions with regulatory cyclins and through phosphorylation of p34 cdc2 itself. p34 cdc2 is defined as a cyclin-dependent kinase since it is inactive unless it is associated with a regulatory cyclin subunit. Furthermore, p34 cdc2 is inhibited by phosphorylation of Tyr-15 and/or Thr-14 (5, 6) but requires phosphorylation of Thr-161 to be activated (7,8). CAK (the p34 cdc2 activating kinase) is responsible for phosphorylation of Thr-161 (9 -11), while the phosphorylation state of Thr-14 and/or Tyr-15 is at least in part controlled by the relative activities of the Wee1 protein kinase (12, 13) and cdc25 protein phosphatase (14,15).Concomitant with the activation of p34 cdc2 at the G 2 -M transition of eukaryotic cells is a massive burst of protein phosphorylation. Many of the events that are associated with entry into mitosis including nuclear envelope breakdown, transcriptional termination, nucleolar disassembly, cytoskeletal reorganization, and chromosome condensation appear to be associated with protein phosphorylation. While it is evident that p34 cdc2 directly phosphorylates a number of proteins at the G 2 -M transition, there are also indications that p34 cdc2 could indirectly regulate phosphorylation events through its phosphorylation of other protein kinases (16 -23).One protein serine/threonine kinase that could be regulated by p34 cdc2 is casein kinase II (CKII), 1 which has been shown to be dramatically phosphorylated in mitotic cells (19 -21). CKII is a tetrameric enzyme composed of two catalytic (␣ and/or ␣Ј-subunits) and two additional subunits (␤ s...