Transforming growth factor beta1 (TGF-β1) suppresses tumor development at early stages of cancer, but enhances tumor invasion and formation of metastasis. TGF-β1-mediated tumor invasion is associated with epithelial to mesenchymal transition (EMT) and matrix proteolysis. The mechanisms of these TGF-β1 responses in normal and tumor cells are not well understood. Recently, we have reported that TGF-β1 increases expression of high-molecular weight tropomyosins (HMW-tropomyosins) and formation of actin stress fibers in normal epithelial cells. The present study investigated the role of tropomyosin in TGF-β1-mediated cell motility and invasion. We found that TGF-β1 restricts motility of normal epithelial cells although it promotes EMT and formation of actin stress fibers and focal adhesions. Cell motility was enhanced by siRNA-mediated suppression of HMW-tropomyosins. TGF-β1 stimulated migration and matrix proteolysis in breast cancer MDA-MB-231 cells that express low levels of HMW-tropomyosins. Tet-Off-regulated expression of HMW-tropomyosin inhibited cell migration and matrix proteolysis without affecting expression of matrix metalloproteinases. Tropomyosin increased cell adhesion to matrix by enhancing actin fibers and focal adhesions. Finally, tropomyosin impaired the ability of tumor cells to form lung metastases in SCID mice. Thus, these results suggest that HMW-tropomyosins are important for TGF-β-mediated control of cell motility and acquisition of the metastatic potential. © 2007 Wiley-Liss, Inc.