IKKbeta {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase beta} is required to activate the transcription factor NF-kappaB, but how IKKbeta itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more 'upstream' protein kinases in some reports, but by autophosphorylation in others. In the present study, we resolve this contro-versy by demonstrating that the activation of IKKbeta induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 [TGFbeta (transforming growth factor beta)-activated kinase-1]-catalysed phosphorylation of Ser177 and, secondly, the IKKbeta-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKbeta to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of linear ubiquitin chains and their interaction with the NEMO (NF-kappaB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKbeta at Ser177 and IKKalpha at Ser176. These findings may be of general significance for the activation of other protein kinases.