In Saccharomyces cerevisiae, the entrance into S phase requires the activation of a specific burst of transcription, which depends on SBF (SCB binding factor, Swi4/Swi6) and MBF (MCB binding factor, Mbp1/Swi6) complexes. CK2 is a pleiotropic kinase involved in several cellular processes, including the regulation of the cell cycle. CK2 is composed of two catalytic subunits (α and α') and two regulatory subunits (β and β'), both of which are required to form the active holoenzyme. Here we investigate the function of the CK2 holoenzyme in Start-specific transcription. The ckb1Δ ckb2Δ mutant strain, bearing deletions of both genes encoding CK2 regulatory subunits, shows a delay of S-phase entrance due to a severe reduction of the expression of SBF- and MBF-dependent genes. This transcriptional defect is caused by an impaired recruitment of Swi6 and Swi4 to G1 gene promoters. Moreover, CK2 α and β' subunits interact with RNA polymerase II, whose binding to G1 promoters is positively regulated by the CK2 holoenzyme. Collectively, these findings suggest a novel role for the CK2 holoenzyme in the activation of G1 transcription.