Signal-induced phosphorylation of IκBα targets this inhibitor of NF-κB for ubiquitination and subsequent degradation, thus allowing NF-κB to enter the nucleus to turn on its target genes. We report here the identification of an IκB–ubiquitin (Ub) ligase complex containing the F-box/WD40-repeat protein, β-TrCP, a vertebrate homolog of Drosophila Slimb. β-TrCP binds to IκBα only when the latter is specifically phosphorylated by an IκB kinase complex. Moreover, immunopurified β-TrCP ubiquitinates phosphorylated IκBα at specific lysines in the presence of Ub-activating (E1) and -conjugating (Ubch5) enzymes. A β-TrCP mutant lacking the F-box inhibits the signal-induced degradation of IκBα and subsequent activation of NF-κB-dependent transcription. Furthermore, Drosophila embryos deficient in slimb fail to activate twist and snail, two genes known to be regulated by the NF-κB homolog, Dorsal. These biochemical and genetic data strongly suggest that Slimb/β-TrCP is the specificity determinant for the signal-induced ubiquitination of IκBα.