The transcriptional activity of NF-κB is stimulated upon phosphorylation of its p65 subunit on serine 276 by protein kinase A (PKA). The transcriptional coactivator CBP/p300 associates with NF-κB p65 through two sites, an N-terminal domain that interacts with the C-terminal region of unphosphorylated p65, and a second domain that only interacts with p65 phosphorylated on serine 276. Accessibility to both sites is blocked in unphosphorylated p65 through an intramolecular masking of the N terminus by the C-terminal region of p65. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal regions of p65 and creates an additional site for interaction with CBP/p300. Therefore, PKA regulates the transcriptional activity of NF-κB by modulating its interaction with CBP/p300.