Casein kinase 2 activity could be resolved into three peaks by chromatography on DEAE-Sepharose. The peak eluted at high salt concentrations (casein kinase 2b) showed molecular and kinetic properties typical of the heterotetramer composed of α-(or α'-) and β-subunits. In contrast, the peak that was eluted at low salt concentrations (casein kinase 2a) contained no β-subunit but a phosphorylatable protein of 49 kDa (pp49), in addition to the α/α'-subunits. The presence of α/α'/α″-subunits in preparations of casein kinases 2a and 2b was confirmed by immunological assays. Casein kinase 2a had low specific activity and a very high apparent K(m) for β-casein. The peak eluted at intermediate ionic strength contained the α/α'-subunits and variable amounts of β-subunit and pp49, and had kinetic properties intermediate between those of casein kinases 2a and 2b. Experiments based on heat inactivation, inhibition by low concentrations of heparin and ability to use GTP as substrate suggested that phosphorylation of pp49 was catalysed by the α/α'-subunits of casein kinase 2. No similarities were observed in the phosphopeptide maps of pp49 and β-subunit. These results show that the α/α'-subunits of rat liver cytosol casein kinase 2 can form complexes not only with the β-subunit but also with pp49, and that the complexes containing pp49 have a reduced affinity for the exogenous protein substrate β-casein.