Protein phosphorylation by the ubiquitous casein kinase II (CKII) is known to be sensitive to naturally occurring polyamines. Using isolated recombinant alpha and beta subunits of the kinase, as well as the alpha 2 beta 2 oligomeric enzyme, it is shown that (i) CKII binds [3H]-spermine with Kds in the micromolar range. (ii) The beta subunit appears mostly responsible for this binding activity. (iii) The isolated alpha (catalytic) subunit is not activated by polyamines. (iv) The polyamine-dependent activation of the oligomeric CKII requires the beta subunit, which appears as a regulatory component in the native kinase. These observations suggest that there may be a functional interaction between polyamines and CKII in living cells, especially in the response to cell growth factors and trophic hormones.