The functions of the upstream stimulatory factors USF1 and USF2 are, like those of other transcription factors, regulated by reversible phosphorylation. Besides many other kinases also protein kinase CK2 phosphorylates USF1 but not USF2. In a yeast-two-hybrid screen, however, the non-catalytic CK2β subunit of CK2 was identified as a binding partner of USF2. This surprising observation prompted us to investigate the CK2/USF interaction in more detail in the present study. By using immunofluorescence analyses as well as co-immunoprecipitations we found that USF1 and USF2 bound to CK2α and CK2β exclusively in the nucleus, though CK2β and to a minor amount CK2α were also present in the cytoplasm. Furthermore, we found that unlike other substrates the phosphorylation of USF1 required the presence of the regulatory CK2β subunit; the catalytic α-subunit of CK2 alone was not able to phosphorylate USF1. Thus, the correct phosphorylation of USF1 is only guaranteed and strictly controlled in particular by nuclear CK2β. Although the data indicated that a nuclear subfraction of CK2 subunits associated with USF proteins, DNA pull down experiments revealed that the CK2 subunits did not co-localize with DNA bound USF proteins indicating that the USF/CK2 interaction has a pre- or post DNA binding function.