Nuclear factor-κB (NFκB) is an inducible transcription factor that plays a key role in regulating the expression of a wide range of immune and inflammatory response genes. The activity of NFκB is controlled at multiple levels, including cytoplasmic retention with inhibitor of κB (IκB) proteins in the basal state. Persistent activation of the transcription factor is seen in numerous chronic inflammatory disease states, and we have previously demonstrated sustained activation of NFκB in human glial cells upon stimulation with interleukin (IL)-1β. In these cells, NFκB retains DNA binding activity for up to 72 h despite the presence of resynthesized IκBα and in the absence of IκBβ. Here we characterized the apparent inability of newly synthesized IκBα to terminate activation of NFκB in glial cells. We showed unexpectedly that newly synthesized IκBα can enter the nucleus, interact with the NFκB subunit p65, and export it to the cytoplasm. However, in vitro analysis of enzyme activity demonstrates that IL-1β causes the long term activation of the IκB kinase complex leading to chronic phosphorylation of the newly synthesized IκBα signal response domain and persistent activation of NFκB. Such sustained activation of NFκB is dependent on the continuous presence and activity of IL-1β. Interestingly, the sustained nature of NFκB activity is promoter type-specific. Chromatin immunoprecipitation studies revealed that p65 is detected at the promoters of both intercellular adhesion molecule-1 and IL-8 1 h following IL-1β stimulation but is only found at the latter at 24 h. The functional significance of this finding is indicated by the transient induction of intercellular adhesion molecule-1 mRNA, but more sustained induction of IL-8 expression, by IL-1β. These studies thus demonstrated that persistent IL-1 signaling causes sustained activation of NFκB in a promoter-specific manner in human glial cells, leading to prolonged induction of selective pro-inflammatory genes. This is likely to make a key contribution to chronic inflammatory conditions of the brain.