A previously described wheat germ protein kinase (Yan, T. F., and Tao, M. (1982) J. Biol. Chem. 257, 7037-7043) was identified unambiguously as CK2 using mass spectrometry. CK2 is a ubiquitous eukaryotic protein kinase that phosphorylates a wide range of substrates. In previous studies, this wheat germ kinase was shown to phosphorylate eIF2␣, eIF3c, and three large subunit (60 S) ribosomal proteins (Browning, K. S., Yan, T. F., Lauer, S. J., Aquino, L. A., Tao, M., and Ravel, J. M. (1985) Plant Physiol. 77, 370 -373). To further characterize the role of CK2 in the regulation of translation initiation, Arabidopsis thaliana catalytic (␣1 and ␣2) and regulatory (1, 2, 3, and 4) subunits of CK2 were cloned and expressed in Escherichia coli. Recombinant A. thaliana CK2 subunits spontaneously dimerize and assemble into holoenzymes in the presence of either CK2␣1 or CK2␣2 and exhibit autophosphorylation. The purified CK2 subunits were used to characterize the properties of the individual subunits and their ability to phosphorylate various plant protein substrates. CK2 was shown to phosphorylate eIF2␣, eIF2, eIF3c, eIF4B, eIF5, and histone deacetylase 2B but did not phosphorylate eIF1, eIF1A, eIF4A, eIF4E, eIF4G, eIFiso4E, or eIFiso4G. Differential phosphorylation was exhibited by CK2 in the presence of various regulatory -subunits. Analysis of A. thaliana mutants either lacking or overexpressing CK2 subunits showed that the amount of eIF2 protein present in extracts was affected, which suggests that CK2 phosphorylation may play a role in eIF2 stability. These results provide evidence for a potential mechanism through which the expression and/or subcellular distribution of CK2 -subunits could participate in the regulation of the initiation of translation and other physiological processes in plants.