Nopp140, a highly phosphorylated nucleolar protein, negatively regulates CK2, a kinase essential for cell proliferation. We quantitatively analyzed the interaction between two subunits of CK2 and Nopp140 and characterized the mechanism by which InsP(6) inhibits the interaction. Nopp140 specifically binds to the catalytic subunit of CK2 (CK2alpha) with a dissociation constant of (K(d)) of 4nM, which interferes with the catalytic activity of CK2. The C-terminal region of Nopp140 is determined as CK2alpha-binding region by a yeast two-hybrid method as well as a direct measurement of the interaction between CK2alpha and deletion mutants of Nopp140. InsP(6) specifically binds to CK2alpha and disrupts the interaction between CK2alpha and Nopp140 with an IC(50) value of 25 microM, thereby attenuating the Nopp140-mediated repression of CK2 activity.