Stimulation of cloned cytotoxic T lymphocytes (CTL) with anti-T-cell receptor (TCR) monoclonal antibody (mAb) in solution resulted in rapid and sustained activation of adhesion to immobilized fibronectin (FN) but did not initiate degranulation. Addition of a second antibody (Ab) to further cross- link the TCR substantially increased the level of adhesion and also activated degranulation, as measured by release of serine esterase, in the presence of immobilized FN but not in its absence. Thus, binding to FN can provide a costimulatory signal to activate degranulation. TCR cross-linking also activated CDS-dependent adhesion to class I, and CD8 provided a costimulatory signal upon binding to class I. However, the requirements for activating adhesion and generating the costimulatory signal differed significantly for FN versus class I ligand, suggesting that these two receptor-ligand systems do not share a common mechanism of action. Co-immobilizing FN and alloantigen resulted in increased serine esterase release in comparison with that stimulated by antigen alone, and required the FN and class I be on the same surface. Peptide and antibody blocking demonstrated that CTL binding to FN, and to vitronectin (VN), was mediated by the αVβ3 vitronectin receptor (VNR). Thus, VNR is activated by a signal from the TCR to mediate adhesion to FN or VN, and delivers a costimulatory signal for degranulation via a different mechanism than costimulation by CD8 binding to class I.