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Interleukin-1 receptor-associated kinase (IRAK-4) is an essential component of the signal transduction complex downstream of the interleukin (IL)-1- and Toll-like receptors. Though regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function has been controversial. In order to investigate the role of IRAK-4 kinase function in vivo, we generated "knock-in" mice where the wild-type IRAK-4 gene is replaced with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase is rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrated lack of cellular responsiveness to stimulation with IL-1beta or Toll-like receptor 4 (TLR4) and TLR7 agonists. IRAK-4 KD cells were severely impaired in NF-kappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. In addition, activation of JNK and p38 was affected in lipopolysaccharide (LPS)-stimulated IRAK-4 KD macrophages. As a consequence, IL-1 receptor/TLR4/TLR7-mediated production of cytokines and chemokines was largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1R-, TLR4-, and TLR7-mediated induction of inflammatory responses.