Plasminogen activator inhibitor type 1 (PAI-1) is induced by many proinflammatory and pro-oxidant factors. Among them, tumor necrosis factor α (TNFα), a pivotal early mediator that regulates and amplifies the development of inflammation, is one of the strongest PAI-1 synthesis activators. Location of the TNFα response element in the PAI-1 promoter is still ambiguous. In this study, we attempted to evaluate the significance of the element located in the 4G/5G site of the PAI-1 promoter in the TNFα stimulation of PAI-1 expression in endothelial cells. PAI-1 expression was monitored at: (a) the level of mRNA using real-time PCR, (b) PAI-1 gene transcription by transfection reporter assays, and (c) protein synthesis using the enzyme immunoassay. NF-κB activity was monitored using the electrophoretic mobility shift assay. Its activity was modified by either antisense oligonucleotides or transfection of endothelial cells with the wild-type or mutated IκBα. We have shown that TNFα-induced expression and gene transcription of PAI-1 involves a regulatory region present in segment −664/−680 of the PAI-1 promoter. This reaction involves the TNFα-induced generation of superoxide leading to activation of NF-κB, and can be abolished by antioxidants and by overexpression of a super-suppressor phosphorylation-resistant IκBα. Stimulation of PAI-1 under these conditions involves the motif of the PAI-1 promoter adjacent to the 4G/5G site, which can directly interact with NF-κB. We show that activation of PAI-1 gene by TNFα and reactive oxygen species is mediated by interaction of NF-κB with the cis-acting element located in the −675 4G/5G insertion/deletion in the PAI-1 promoter.