The proteolytic degradation of the inhibitory protein MAD3/IκBα in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-κB. Analysis of the expression of human IκBα protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous IκBα is degraded in response to inducers of NF-κB activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-nor-leucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyper-phosphorylated form of the human IκBα protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of IκBα. Our results show that deletion of the C terminus of the IκBα molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of IκBα maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysac-charide induces a kinase activity which phosphorylates serines 32 and 36 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-κB to translocate to the nucleus and to activate gene expression.