The -300 region of the interleukin 1β (IL-1β) promoter contains a functional NF-κB binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-κB site alone interacted with NF-κB proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-κB protein-DNA complex formation was dissociated in vitro by the addition of recombinant IκBα. Mutation of the NF-κB site in the context of the IL-1β promoter reduced the responsiveness of the IL-1β promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1β. A 4.4-kb IL-1β promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-κB subunits compared with a mutated IL-1β promoter fragment. When multiple copies of the IL-1β NF-κB site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1β NF-κB site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-κB binding site in the IL-1β promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-κB binding activity. Thus, the IL-1β gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-κB/rel family of transcription factors.