Thrombin is a coagulation system protease that also serves as a potent stimulator of gene expression in several cell types, including endothelial cells (EC). We and others have previously demonstrated that the transcription of platelet-derived growth factor (PDGF) B-chain (c-sis) by EC is stimulated severalfold by thrombin. Here we examine the molecular mechanism of this regulatory process using bovine aortic EC transiently transfected with a vector containing the chloramphenicol acetyltransferase (CAT) gene under the control of a 400-base pair fragment of the human PDGF B-chain promoter. Thrombin treatment of these cells caused a severalfold increase in CAT expression. Deletion analysis and site-directed mutagenesis revealed that the region spanning nucleotides -61 to -53 from the transcription initiation site (referred to as the thrombin response, or ThR, region) was critical for the transcriptional response to thrombin. Electrophoretic mobility shift assays with an oligonucleotide corresponding to the region -64 to -44, which contained the ThR region, led to the identification of a thrombin-inducible nuclear factor (TINF) in extracts from thrombin-treated, but not control, EC. TINF was formed as early as 40 min post-thrombin treatment, persisted for at least 7 h, but was no longer present after 24 h. TINF appeared in the absence of de novo protein synthesis. The ThR region consists of a repeat of a CCACCC element in an ABBA configuration, which, based on mutation analysis and transfection assays, appears to be critical in mediating thrombin stimulation of the PDGF B-chain gene. The conservation of the ThR region in the promoter of the PDGF B-chain among three species (human, feline, and murine) further supports the importance of this region as a cis-acting regulatory element.