Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IkappaBzeta (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IkappaB family member Bcl-3 (refs 1-6). Transcription of IkappaBzeta is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IkappaBzeta is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IkappaBzeta-deficient cells show severe impairment of IL-6 production in response to a variety of TLR ligands as well as IL-1, but not in response to tumour-necrosis factor-alpha. Endogenous IkappaBzeta specifically associates with the p50 subunit of NF-kappaB, and is recruited to the NF-kappaB binding site of the IL-6 promoter on stimulation. Moreover, NF-kappaB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IkappaBzeta-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IkappaBzeta-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IkappaBzeta occurs earlier than transcription of these genes, some TLR/IL-1R-mediated responses may be regulated in a gene expression process of at least two steps that requires inducible IkappaBzeta.