The beta-subunit of eukaryotic translation initiation factor eIF2 is a substrate and a partner for protein kinase CK2. Surface plasmon resonance analysis shows that the truncated form corresponding to residues 138-333 of eIF2beta (eIF2beta-CT) interacts with CK2beta as efficiently as full length eIF2beta, whereas the form corresponding to residues 1-137, which contains the CK2 phosphorylation sites, (eIF2beta-NT) does not bind. The use of different mutants and truncated forms of CK2alpha allowed us to map the basic segment K74-K83 at the beginning of helix alphaC and residues R191R195K198 in the p + 1 loop as the main determinants for the binding to eIF2beta-CT of either the isolated CK2alpha subunit or the CK2 holoenzyme. The presence of eIF2beta-CT stimulated the activity of CK2alpha towards the RRRAADSDDDDD peptide substrate; effect that was not observed with the CK2a K74-77A whose ability to bind to eIF2beta-CT is severely impaired. Gel filtration analysis confirmed the ability of CK2alpha to form complexes with eIF2beta-CT, and the contribution of the basic cluster in CK2alpha (K74-K77) in this association.