NF-κB is an important transcription factor that controls the inducible expression of more than hundred genes associated with innate and adaptive immunity, inflammation, and apoptosis. The proper regulation of these genes is critical for maintenance of cellular homeostasis. Regulation of NF-κB occurs primarily in the cytoplasm through its association with a member of the IκB family of inhibitor proteins. In resting cells, IκB binds to NF-κB and retains it in the cytoplasm as an inactive complex. This is accomplished by sequestration of the nuclear localization sequence (NLS) of NF-κB and inherent potential of IκB proteins to be exported from the nucleus. In cells that have been stimulated by stress or inflammatory signals, the IκB inhibitor becomes phosphorylated by the IκB Kinase complex (IKK) and is quickly degraded via the ubiquitin-dependent 26 S Proteasome pathway. NF-κB then enters the nucleus and activates transcription of target genes. Previous studies showed that the IκB protein known as IκBβ displays high binding affinity for NF-κB and can disrupt pre-formed NF-κB:DNA complexes in vitro and in cells. This is true of other κB proteins such as IκBα and IκBe. However, IκBβ has also been reported to form a ternary complex with NF-κB on DNA. The ability of IκBβ to either disrupt or stabilize NF-κB:DNA complexes could explain why IκBβ regulates a persistent NF-κB response while other IκB proteins control NF-κB activity transiently. We proposed that post-translational modification of newly synthesized IκBβ might dictate the effect of IκBβ on DNA binding by NF-κB. Specifically, we investigated the potential of a specific serine, murine IκBβ residue Ser346, to become phosphorylated and the effects of that modification on the interaction of IκBβ and NF-κB.