eIF2 (eukaryotic translation-initiation factor 2) is a substrate and an interacting partner for CK2 (protein kinase CK2). Co-immuno-precipitation of CK2 with eIF2beta has now been observed in HeLa cells, overexpressing haemagglutinin-tagged human recombinant eIF2beta. A direct association between His6-tagged human recombinant forms of eIF2beta subunit and both the catalytic (CK2alpha) and the regulatory (CK2beta) subunits of CK2 has also been shown by using different techniques. Surface plasmon resonance analysis indicated a high affinity in the interaction between eIF2beta and CK2alpha, whereas the affinity for the association with CK2beta is much lower. Free CK2alpha is unable to phosphorylate eIF2beta, whereas up to 1.2 mol of phosphate/mol of eIF2beta was incorporated by the reconstituted CK2 holoenzyme. The N-terminal third part of eIF2beta is dispensable for binding to either CK2alpha or CK2beta, although it contains the phosphorylation sites for CK2. The remaining central/C-terminal part of eIF2beta is not phosphorylated by CK2, but is sufficient for binding to both CK2 subunits. The presence of eIF2beta inhibited CK2alpha activity on calmodulin and beta-casein, but it had a minor effect on that of the reconstituted CK2 holoenzyme. The truncated forms corresponding to the N-terminal or central/C-terminal regions of eIF2beta were much less inhibitory than the intact subunit. The results demonstrate that the ability to associate with CK2 subunits and to serve as a CK2 substrate are confined to different regions in eIF2beta and that it may act as an inhibitor on CK2alpha.