To assess the functional role of the four conserved cysteinyl residues in the regulatory beta-subunit of protein kinase CK2, the effect of pCMB and other reagents of sulfhydryl groups has been investigated. The pCMB-treated beta-subunit has lost its ability to form either homodimers or regular alpha(2)beta(2) heterotetramers with the catalytic subunit. It also fails to increase catalytic activity toward peptide substrates and to mediate the stimulatory effect of polylysine, The pCMB-treated beta-subunit, however, is still able to prevent calmodulin phosphorylation and to physically interact with the alpha-subunit to form inactive complexes whose sedimentation coefficient is lower than that of CK2 holoenzyme, These inactive complexes upon treatment with reducing agents like DTT are converted into a fully active heterotetrameric holoenzyme.