The alpha and beta subunits of casein kinase II are dramatically phosphorylated in cells that are arrested in mitosis (Litchfield, D. W., Lüscher, B., Lozeman, F. J., Eisenman, R. N., and Krebs, E.G. (1992) J. Biol. Chem. 267, 13943-13951). Comparative phosphopeptide mapping experiments indicated that the mitotic phosphorylation sites on the alpha subunit of casein kinase II can be phosphorylated in vitro by p34cdc2. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the alpha subunit is phosphorylated by p34cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of alpha. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34cdc2 phosphorylation. Synthetic peptides corresponding to regions of alpha that contain each of these residues are phosphorylated by p34cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of alpha 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 p34cdc2 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 alpha subunit of casein kinase II that are phosphorylated in mitotic cells.