The kinases and regulatory proteins that convey signals initiated by transforming growth factor-β (TGF-β) to the nucleus are poorly characterized. To study the role of the extracellular signal-regulated kinase (ERK) pathway in this process, we transiently transfected NIH 3T3 fibroblasts with TGF-β-responsive luciferase reporter genes and expression vectors designed to interrupt this kinase cascade. Mitogen-activated protein (MAP) kinase phosphatase-1 and a dominant negative MAP/ERK kinase 1 mutant reduced stimulation of plasminogen activator inhibitor-1 (PAI-1) promoter activity by TGF-β1 from 11.5- to 4-fold and 4.9-fold, respectively. Similar results were observed with the type I collagen promoters. TGF-β1 increased ERK1 activity 4.5-fold at 5 min and 3.1-fold at 3 h, while Jun kinase and p38 activity were not affected. Cotransfection of a dominant negative mutant of the small G protein, Rac, but not dominant negative Ras, Cdc42, or Rho mutants, reduced the effects of TGF-β1 on the PAI-1 promoter by approximately half. In support of a role for Rac in signaling by TGF-β, GTP binding to Rac was increased 3.7-fold following exposure of NIH 3T3 cells to TGF-β1 for 3 min. These findings indicate that TGF-β1 modulates gene expression partly through ERK and Rac in NIH 3T3 cells.