We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose-and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-κB site (− 533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-κB site (− 223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-κB site is critical for thrombin inducibility. NF-κB-directed luciferase activity increased∼ 3-fold when cells transfected with the plasmid pNF-κBLUC containing five copies of consensus NF-κB site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-κB was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-κBp65 (Rel A) to downstream NF-κB site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH 2 terminus of proteolytically activated receptor-1, mimicked thrombin’s action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-κBp65 binding to the downstream NF-κB site of ICAM-1 promoter after proteolytically activated receptor-1 activation.