To define the cis-acting elements that regulate LPS-stimulated IL-1β gene transcription, we analyzed the murine IL-1β gene by digestion with DNase I. At least two hypersensitive sites were located between 2200 and 2600 bp upstream of the transcription start site in mononuclear phagocytes, but not in an IL-1 nonproducing immature T cell line. Specific DNA sequences required for LPS induction of IL-1β gene expression were identified within the DNase I hypersensitive (DH) region using transfection of reporter constructs that contained portions of the IL-1β 5'-flanking region. Two specific DNA sequences were targets for nuclear factor binding as assessed with use of electrophoretic mobility shift analysis (EMSA). One site contained a consensus sequence for NFIL-6 binding. Base substitutions within this NFIL-6 site resulted in virtual elimination of LPS-induced IL-1β gene transcription. Introduction of multimers of the NFIL-6-like sequence immediately 5' to homologous or heterologous promoters conferred LPS-induced transcription, indicating that this NFIL-6-like consensus site was a transcriptional activator. Anti-C/EBPβ (NFIL-6) and anti-C/EBPδ (NFIL-6β) Abs identified both of these proteins in complexes formed between the NFIL- 6-like element and mononuclear cell nuclear extracts. C/EBPδ (NFIL-6β) was not detected in complexes utilizing extracts from the IL-1 nonproducing T cell line. These data are consistent with the requirement for C/EBPβ (NFIL- 6) and C/EBPδ (NFIL-6β) in the activation of murine IL-1β gene expression by endotoxin.