Members of the IKK {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase} family play a central role in innate immunity by inducing NF-kappaB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKK/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-kappaB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKbeta, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKK and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFalpha-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-kappaB essential modulator), which is associated with reduced IKKalpha/beta activity and NF-kappaB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IkappaBalpha, p105, RelA (IKKalpha and IKKbeta) and IRF3 (IKK and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.