The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-κB family members interact to regulate the human TNF-α gene. A 120-bp TNF-α promoter-reporter, possessing binding sites for NF-κB (κB3), C/EBPβ (CCAAT/enhancer binding protein β), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-κB p65, and c-Jun, but not C/EBPβ, suppressed (p < 0.05- 0.001) LPS-induced TNF-α secretion in primary human macrophages. Following LPS stimulation, NF-κB p50/p65 heterodimers bound to the κB3 site and c-Jun to the -103 AP-1 site of the TNF-α promoter. By transient transfection, NF- κB p65 and p50 synergistically activated the TNF-α promoter. In contrast, no synergy was observed between NF-κB p65, with or without NF-κB p50, and c-Jun or C/EBPβ, even in the presence of the coactivator p300. The contribution of the upstream κB binding sites was also examined. Following LPS stimulation, the κB1 site bound both NF-κB p50/p65 heterodimers and p50 homodimers. The binding by NF-κB p50 homodimers to the κB1, but not to the κB3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent κB3 and AP-1 sites in the human TNF-α promoter contribute independently to LPS-induced activation. Although both the κB1 and κB3 sites bound transcriptionally active NF-κB p50/p65 heterodimers, only the κB1 site contributed to down-regulation by NF-κB p50 homodimers.