The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin alpha(5)beta(1). In this work, we determined whether the activity directed by the alpha(5) gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/alpha(5) promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the alpha(5) FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3' half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its alpha(5)beta(1) integrin activates a signal transduction pathway that results in the transcriptional activation of the alpha(5) gene likely through altering the phosphorylation state of Sp1.