Cell-cell and cell-extracellular matrix (ECM) binding mediated by integrin molecules has been implicated in lymphocyte migration and adhesion. We describe here that ECM binding triggers antigen-independent activation of T cell functions. Fibronectin and vitronectin, when coated on plates, not only acted synergistically on anti-CD3-induced serine esterase release in a murine cytotoxic T lymphocyte clone and interleukin 2 production in a murine helper T cell hybridoma but also could trigger these responses alone. All these stimulatory effects of ECM were abrogated by a monoclonal antibody which reacts with a unique very late activation antigen-like integrin and this monoclonal antibody, when coated on plates, exhibited a similar synergistic effect to that of ECM proteins. Therefore, ECM receptors expressed on activated T cells appear to play an important role in triggering T cell effector functions in localized tissues abundant in ECM proteins.