EGR-1, a transcription factor with important functions in the regulation of growth and differentiation, is highly expressed in brain. Previous studies have shown that EGR-1 suppresses the transformed phenotype. However, the expression and role of EGR-1 in human glioblastoma cells are not yet determined. In this study, we found that the basal expression of the EGR-1 protein is undetectable, but is inducible in four human glioblastoma cell lines. To determine EGR-1 functions, we re-expressed EGR-1 in human glioblastoma U251 cells and found that the secretion of transforming growth factor-beta1 (TGF-beta1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin (FN) was greatly enhanced. Addition of anti-TGF-beta antibodies completely inhibited the secretion of PAI-1, but had little effect on secretion of FN, indicating that PAI-1 is under the control of EGR-1-induced TGF-beta1. An examination of the promoter of the FN gene revealed two EGR-1-binding sites between positions -75 and -52 and positions -4 and +14 that specifically bound EGR-1 in gel mobility shift experiments. Utilizing wild-type and mutant FN promoter/luciferase reporter genes, we demonstrated that EGR-1 positively regulated the activity of the FN gene. In addition, cell adhesion and migration were greatly increased in the EGR-1-expressing cells, and adhesion was reversed by addition of RGD-containing peptides. These results suggest that EGR-1 may regulate cell interaction with the extracellular matrix by coordinated induction of TGF-beta1, FN, and PAI-1 in human glioblastoma cells.