Mammalian RNA polymerase I (Pol 1) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol 10 isoform and not with Pol fix. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol I beta-associated CK2 can phosphorylate topoisomerase II alpha in Pol I beta, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAF(I)110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAF(I)110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.