Lung injury, characterized by the flooding of interstitial and alveolar spaces with serum proteins, induces the expression of fibronectin (FN). This cell-adhesive extracellular matrix (ECM) glycoprotein is believed to modulate inflammation and wound repair. Murine NIH/3T3 fibroblasts transfected with a 1.2-kb human FN promoter-reporter gene were studied to gain insight into the mechanisms involved in the induction of FN by serum. Transcription of the FN gene, followed by FN protein production, was enhanced by 10% fetal bovine serum. This effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinases. ECMs typically found in injured tissues (i.e., type I collagen, fibrin, and FN) had no effect. Conversely, disruption of actin microfilaments inhibited, whereas disruption of microtubular assembly enhanced, the serum-induced FN response. The stimulatory effects of serum and microtubular disruption on FN gene transcription were related to increased DNA binding of the transcription factor cAMP response element binding protein. The data suggest that regulation of serum-induced FN expression in fibroblasts is dependent on protein kinases and on cytoskeletal integrity.