Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.