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. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. 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 (-2280 to -2275) in the chimeric IL-1β promoter -4093 +I 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 -2281 to -2271, 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.