Multiple transforming growth factor-ß (TGF-ß) responsive elements have been identified within the 5’-flanking region of the plasminogen activator inhibitor type-1 (PAI-1) gene. This study was designed to characterize the major TGF-ß responsive element (-804 to -546). DNA footprint assays showed that the region of protein contact (-726 to -703) did not include consensus sequences for any known transacting factors. The results of UV cross-linking and Southwestern blot experiments showed that a protein of M, 100,OOO specifically binds to the TGH-ß responsive element and that this protein undergoes post-transcriptional activation within 5 min after stimulation of Hep G2 cells by TGF-ß resulting in a marked increase in affinity for the target DNA sequence. These results show that stimulation of Hep G2 cells with TGF-ß increases the affinity of a novel pr 100-kDa protein for the major TGF-ß responsive element within the PAI-1 gene.