Regulatory elements important for transcription of the murine interleukin-1β (IL-1β) gene lie within a DNase I-hypersensitive region located> 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1β promoter–chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264. 7. Electro-phoretic mobility shift analysis of the 3′ portion (22315 to 22106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions 22285 and 22256. Competitive inhibition studies localized the binding site to a 15-bp sequence between 22285 and 22271. Nuclear factor binding was lost by mutation of the 6-bp sequence from 22280 to 22275. The specific retarded complex formed with RAW264. 7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1β RNA. Mutation of the 6-bp sequence (22280 to 22275) in the chimeric IL-1β promoter 24093 1I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264. 7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5′ of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from 22281 to 22271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1β-upstream nuclear factor 1 (IL1β-UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1β-UNF1-specific binding factor approximately 85 to 90 kDa in size.