Human intercellular adhesion molecule-1 (ICAM-1) plays an important role in immune responses as the major specific ligand for the beta2-integrins LFA-1 and Mac-1. During the inflammatory process, ICAM-1 expression is stimulated by various proinflammatory cytokines. We have examined the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by tumor necrosis factor-alpha (TNF-alpha) and by the nuclear factor-kappaB (NF-kappaB) family of transcription factors in the Ad5-transformed human embryonal kidney cell line 293. A proximal site (5'-TTGGAAATTCC-3') mapping at position -228 from the ATG and known to mediate TNF-alpha responsiveness in endothelial cells is also critical for TNF-alpha responsiveness in 293 cells. However, unlike endothelial cells, electrophoretic mobility shift assays, using whole-cell extracts prepared from TNF-alpha-treated cells, showed that TNF-alpha induces the formation of a specific kappaB binding complex, mainly composed of NF-kappaB subunits RelA and c-Rel. Electrophoretic mobility shift assays done with 293 cells transfected with p50, p65, or both subunits showed that p50 only has a weak ability to bind the proximal ICAM-1 NF-kappaB site. Another element exhibiting sequence homology with NF-kappaB binding sites and located at position -540 relative to the mRNA cap site was found to be involved in the basal activity of the ICAM-1 promoter, is not required for TNF-alpha responsiveness, and does not bind NF-kappaB subunits. Whereas transactivation of the ICAM-1 promoter by p65 requires the proximal NF-kappaB site, deletion mutant analysis showed that p50 and, to a greater extent, p52 transactivate reporter plasmids lacking NF-kappaB sites, suggesting the presence of other p50/p52 responsive element(s).