The protein kinase CK2 (former name: “casein kinase 2”) predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) and two noncatalytic subunits (CK2β). The CK2β subunits form a stable dimer to which the CK2α monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2β is no on-switch of CK2α; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2α and CK2β that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2α on CK2β with an upshift of the CK2α melting temperature of more than 9°. Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2α and CK2β is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2β construct to 2.8 Å resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2β conformation is largely conserved upon association with CK2α, whereas the latter undergoes significant structural adaptations of its backbone.