Abstract Transforming growth factor-β1 (TGF-β1) transcriptionally regulates the expression of genes that encode specific proteins (e.g., plasminogen activator inhibitor-1; PAI-1) important in stromal remodeling and cellular invasion. Definition of molecular events underlying TGF-β1-initiated PAI-1 transcription, therefore, may lead to the identification of new therapeutic targets for diseases associated with elevated PAI-1 synthesis (e.g., tissue fibrosis, vascular disorders, tumor progression). An intact upstream stimulatory factor (USF)-binding E box motif (5′- −165CACGTG −160-3′) at the HRE-2 site in the rat PAI-1 gene was required for PAI-1 transcription in TGF-β1-treated cells. Mutation of the CA dinucleotide to TC at position −165/−164 in a reporter construct driven by 764 bp of PAI-1 promoter sequence decreased TGF-β1-dependent CAT activity by >80% indicating the necessity for a consensus hexanucleotide E box motif in induced expression. The same CA → TC substitution eliminated USF binding to an 18-bp HRE-2 DNA target highlighting the importance of site occupancy to transcriptional activation. Transfection of a dominant-negative USF construct, moreover, completely inhibited formation of USF/HRE-2 probe complexes, attenuated PAI-1 promoter-driven luciferase activity and reduced the response of the endogenous PAI-1 gene to TGF-β1 (to that approximating quiescent controls). Maximal immediate-early PAI-1 induction upon exposure to TGF-β1 required EGFR, p21 ras , MEK and pp60 c- src signaling as pharmacologic or dominant-negative inhibition of any of the four intermediates (EGFR, p21 ras , MEK, pp60 c- src ) virtually eliminated TGF-β1-augmented PAI-1 levels. U0126 titering experiments, furthermore, revealed that the same MEK inhibitor concentration that blocked the TGF-β1 increase in ERK1/2 phosphorylation (20 μM) also effectively attenuated the PAI-1 inductive response suggesting a requirement for stimulated ERK signaling in TGF-β1-mediated PAI-1 expression. These data suggest a model whereby TGF-β1 activates a complex signaling cascade to affect PAI-1 gene control and involves USF occupancy of a critical E box motif at the HRE-2 site in the PAI-1 gene.