Activation of the transcription factor NF-κB in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-κB inhibitor, IκBα. Here, we show the in vitro reconstitution of the phosphorylation-dependent ubiquitination of IκBα with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quaternary SCF^HOS–ROC1 holoenzyme (with Skp1 and the β-TRCP homolog HOS). SCF^HOS–ROC1 binds IKKβ-phosphorylated IκBα and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization.