SMAD3 is a transcription factor that mediates TGF-beta1 signaling and is known to be important in many of the cellular processes that regulate fibrosis and inflammation. Although several studies have examined SMAD3 activation, little is known about the control of SMAD3 expression. It is well established that the mitogen-activated protein kinase (MAPK) pathway is responsive to TGF-beta1 stimulation and coordinates with SMAD signaling in many cases; therefore, the hypothesis of this study is that the MAPK pathway will be involved in the regulation of SMAD3 expression. Using a SMAD3 promoter construct, we demonstrate that inhibition of either c-Jun-N-terminal kinase (JNK) or p38 activity has little effect on SMAD3 promoter function. Inhibition of mitogen-activated protein kinase kinase-1 (MEK1) with either PD98059 or UO126, however, results in a substantial dose-dependent inhibition of SMAD3 promoter activity. Further studies confirm that promoter activity correlates with protein expression by demonstrating reduced SMAD3 protein expression in A549 cells and airway smooth muscle cells after treatment with MEK1 inhibitors. Positive regulation of SMAD3 expression is also demonstrated by expression of a constitutively active (ca)-MEK1 construct, where the presence of ca-MEK1 resulted in increased SMAD3 protein expression. These data lead to the conclusion that MEK1 is an important regulator of SMAD3 expression.