Extracellular stimuli that activate the transcription factor NF-κB cause rapid phosphorylation of the IκBα inhibitor, which retains NF-κB in the cytoplasm of nonstimulated cells. Phosphorylation of IκBα is followed by its rapid degradation, the inhibition of which prevents NF-κB activation. To determine the relationship between these events, we mapped the inducible phosphorylation sites of IκBα. We found that two residues, serines 32 and 36, were phosphorylated in response to either tumor necrosis factor, interleukin-1, or phorbol ester. Substitution of either serine blocks or slows down induction of IκBα degradation. Substitutions of the homologous sites in IκBβ, serines 19 and 23, also prevent inducible IκBβ degradation. We suggest that activation of a single IκB kinase or closely related IκB kinases is the first critical step in NF-κB activation. Once phosphorylated, IκB is ubiquitinated. Unlike wild-type IκBα, the phosphorylation-defective mutants do not undergo inducible polyubiquitination. As substitution of a conserved lysine residue slows down the ubiquitination and degradation of IκBα without affecting its phosphorylation, polyubiquitination is required for inducible IκB degradation.