Protein kinase CK2 is an ubiquitous Ser/Thr kinase essential for cell growth. We have used the highly synchronizable tobacco BY-2 cell line to investigate whether CK2 activity and expression are regulated in a cell cycle phase-dependent manner in higher plants. Specific cDNA probes for tobacco CK2α and β subunits, respectively, and polyclonal antibodies recognising α and β subunits separately, were obtained to determine mRNA and protein levels of both subunits. Our results show that CK2 activity oscillates throughout the cell cycle, peaking at G1/S and M phases, due to a posttranslational regulation of the tetrameric enzyme. Additional levels of control of CK2 expression operate in relation to the proliferative state of the cells, including differential accumulation of α and β transcripts and posttranscriptional regulation of protein levels (β subunit). Moreover, in vivo inhibition of CK2 activity corroborates the requirement of the functional CK2 to progress through the cell division cycle, and suggests that CK2 might play an important role at the G2/M checkpoint.