Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1. A DNase I-hypersensitive site in the 5′ proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-α treatment, suggesting the presence of a TNF-α-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is sufficient to confer TNF-α inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (–129 to –110 and –99 to –80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-κΒ binding sites (site 1, –126; site 2, –116; and site 3, –94). All three sites can be bound by NF-κΒ when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-κΒ proteins bound to multiple regulatory elements.