Transforming growth factor beta (TGFbeta) plays an important role in animal development and many cellular processes. A variety of cellular functions that are required for tumor metastasis are controlled by integrins, a family of cell adhesion receptors. Overexpression of alphaVbeta6 integrin is associated with lymph node metastasis of gastric carcinomas. It has been demonstrated that a full TGFbeta1 signal requires both alphaVbeta6 integrin and SMAD pathway. TGFbeta1 binds to alphaVbeta6 via the DLXXL motif, a freely accessible amino acid sequence in the mature form of TGFbeta1. Binding of mature TGFbeta1 to alphaVbeta6 leads to immobilization and tyrosine phosphorylation of proteins, which are associated with focal adhesions, a hallmark of integrin-mediated signal transduction. Here, we show that binding of mature TGFbeta1 recruits the mitogen-activated protein kinase kinase kinase 1 (MEKK1), a mediator of c-Jun activation, and the extracellular signaling-regulated kinase-1 (Erk1) to focal adhesions. In addition, the p21-activated kinase 1 (PAK1) is associated with focal adhesions and differentially phosphorylated upon TGFbeta1 stimulation. We conclude that TGFbeta1 activates c-Jun via the MEKK1/p38 MAP kinase pathway and influences cytoskeletal organization. These finding may provide a link between TGFbeta1 and the metastatic behavior of cancers.