Stimulation of endothelial cells by cytokines and bacterial lipopolysaccharide leads to activation of the transcription factor NF-kappa B. NF-kappa B in turn regulates the expression of several genes involved in the inflammatory reaction, including cell adhesion molecules, interleukins, and transcription factors. One of these induced genes encodes an inhibitor of NF-kappa B, ECI-6/I kappa B alpha, that contains in its 5' regulatory region six consensus binding sites for NF-kappa B. We demonstrate here that these sites display striking differences in their ability in vitro to bind to various NF-kappa B subunits. In vivo, all six sites contribute, though to varying degrees, to transcription from the ECI-6/I kappa B alpha promoter, as demonstrated by deletion and mutation analysis. Among the NF-kappa B subunits tested p65, the p65/p50 heterodimer and, to a lesser extent, c-Rel, are able to activate transcription, whereas p50 or p50/Re1B were inactive. Since many genes regulated by NF-kappa B contain only one or two DNA-binding sites for this transcription factor, the presence of six functional NF-kappa B-binding sites in the ECI-6/I kappa B alpha promoter represents a unique feature of this gene.