Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (alpha and/or alpha') subunits and two regulatory (beta) subunits. Because CK2beta is synthesized in excess of CK2alpha, we hypothesized that formation of CK2beta homodimers precedes the incorporation of the catalytic subunits of CK2 into complexes. To test this hypothesis, we cotransfected cells with two epitope-tagged variants of CK2beta. The results of these cotransfection studies demonstrate that interactions between two CK2beta subunits take place in the absence of CK2alpha. Together with results from previous biosynthetic labeling studies, these results suggest that formation of CK2beta homodimers occurs before incorporation of catalytic subunits of CK2 into CK2 complexes. We also cotransfected Cos-7 cells with a deletion fragment of CK2beta (i.e. Myc-beta1-166) together with full-length hemagglutinin (HA)-tagged CK2beta and/or CK2alpha'. Although complexes between Myc-beta1-166 and HA-beta were readily detected, we obtained no evidence of direct interactions between Myc-beta1-166 and HA-CK2alpha'. These results suggest that residues within the N-terminal 166 amino acids of CK2beta are sufficient for interactions between CK2beta subunits, whereas the C-terminal domain of CK2beta is required for complex formation with the catalytic subunits of CK2. Finally, we observed that expression of full-length HA-beta promotes phosphorylation of Myc-beta1-166 by HA-CK2alpha'.