Rel proteins are important intracellular mediators of cytokine-induced signal transduction. To understand how cytokines affect different cell populations in the brain, we have characterized Rel activation in astrocytes. A RelA homodimer is uniquely activated in cytokine-stimulated astrocytes. Cytokine-dependent phosphorylation of the RelA inhibitor MAD-3 occurred on discrete peptides prior to its dissociation from RelA. A transient hyperphosphorylation of RelA was also induced. Antioxidant treatment inhibited both RelA activation and phosphorylation of the RelA.MAD-3 complex. These results demonstrate that cytokine-dependent activation of the RelA homodimer involves phosphorylation of both RelA and its associated inhibitor. The sole activation of a RelA homodimer suggests that cytokines will activate a unique set of Rel-regulated genes in astrocytes.