Although the involvement of transforming growth factor-β (TGF-β) in inflammatory reactions has been extensively studied, its mode of action in the context of the extracellular matrix (ECM) is still not fully understood. We undertook this study in an attempt to reveal the putative roles of TGF-β in T-cell adhesion and migration. We found that a 60-min treatment of T cells with TGF-β regulates T-cell adhesion to fibronectin (FN), a prototype cell adhesion protein of the ECM, depending on the presence of other activators. At 5 pg/ml to 1 ng/ml, TGF-β alone induced T-cell adhesion to FN in an integrin α4/β1- and integrin α5/β1-dependent manner. TGF-β also attenuated T-cell migration on the stromal cell-derived factor (SDF)-1α gradients. These effects of TGF-β were not accompanied by alteration in the expression of very-late activation antigen type 4 (VLA-4) and VLA-5, nor were they mediated by the cyclo-oxygenase pathway. The cellular mechanism underlying the adhesion-regulating activities of TGF-β involves adhesion-associated cytoskeletal elements. TGF-β induced the phosphorylation of focal adhesion kinase Pyk2, but not extracellular signal-regulated kinase (ERK), and this effect was markedly increased in the presence of immobilized FN, suggesting a collaborative role for FN-specific integrins. Indeed, TGF-β-induced Pyk2 phosphorylation was inhibited by monoclonal antibodies against VLA-4, VLA-5 and CD29. Thus, TGF-β, which may appear at extravascular sites during inflammation, affects the adhesion of T cells to ECM glycoproteins and their migration by its ability to differentially induce or inhibit the phosphorylation of Pyk2.