Protein kinase CK2 is a ubiquitous eukaryotic protein kinase composed of two catalytic subunits, alpha and/or alpha', and two regulatory subunits, beta. In order to define similarities and dissimilarities between the alpha and alpha' catalytic subunits, which might account for their particular cellular functions, different forms of the enzyme were expressed in Sf9 cells and their properties determined. Both catalytic subunits were expressed separately, and also along with the regulatory beta subunit, in order to obtain free alpha and alpha', as well as alpha2beta2 and alpha'2beta2 forms. Our results confirm that the beta subunit acts to stabilize the alpha and alpha' subunits and also influences the substrate specificity and kinetic properties of the enzyme. Although significant differences between the specificities of holoenzymes alpha2beta2 and alpha'2beta2 as determined using a number of substrates were not detected, autophosphorylation studies on alpha2beta2 and alpha'2beta2 revealed significant differences in this property. The regulatory subunit beta was phosphorylated less rapidly by the alpha' subunit than by the alpha subunit, and the extent of phosphorylation of beta by alpha was also greater than that of beta by alpha'. It was also noted that the thermo-stability and the extent of its activation by NaCl were greater for alpha2beta2 than for alpha'2beta2. These different properties may relate to distinct functions of the two form of CK2.