Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.