Nuclear factor kappa B (NF-kappaB) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappaB is present in the cytosol where it is complexed to its inhibitor IkappaB. Activation of NF-kappaB depends on the signal-induced phosphorylation of IkappaB by specific IkappaB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IkappaBalpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IkappaB kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1), the IkappaB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappaB-related and IkappaB-related proteins: RelA, RelB, cRel, p100, p105, Ikappa Balpha, Ikappa Bbeta and Ikappa Bepsilon. The existence of stable kinase-substrate complexes, the presence of all three known IkappaB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating Ikappa Balpha-, Ikappa Bbeta- and Ikappa Bepsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappaB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of Ikappa Balpha, we observed efficient phosphorylation of serine 32 in Ikappa Balpha by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.