NF-kappa B, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, I kappa Bs(1-3). It, activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pI kappa B alpha-ubiquitin ligase (pI kappa B alpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappa B inhibitor pI kappa B alpha. Taking advantage of its high affinity to pI kappa B alpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pI kappa B alpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RS(I kappa B) (pI kappa B alpha-E3 receptor subunit), binds specifically to pI kappa B alpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, El and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(I kappa B), which tightly binds pI kappa B alpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pI kappa B alpha and consequently NF-kappa B activation. E3RS(I kappa B) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.