The nuclear activity of Rel/NFkappaB transcription factors is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called IkappaBalpha. IkappaBalpha is rapidly phosphorylated and degraded in response to the stimulation through tumor necrosis factor alpha (TNFalpha) receptor, interleukin-1 receptor or CD40. To explore the molecular mechanisms of signal-induced depletion of IkappaBalpha, we have delineated the domain in IkappaBalpha that is required for TNFalpha-induced phosphorylation and rapid degradation of IkappaBalpha. In contrast to the previous reports, the PEST-like sequences, which are present in the carboxyl-terminal region of IkappaBalpha, are demonstrated here to be dispensable for TNFalpha-induced degradation but could be required for signal-independent degradation, as in the case of Cactus, Drosophila homologue of IkappaB. Furthermore, the ankyrin repeats, which are essential for forming a complex with Rel and RelA, are required for TNFalpha-induced degradation suggesting that the putative IkappaB protease could interact with IkappaBalpha in complex with RelA or could recognize the structure of ankyrin repeats. Our data also indicate that neither the ankyrin repeats nor the PEST-like sequences, are essential for TNFalpha-induced phosphorylation.