The relationship between the T cell receptor (TcR) for antigen (Ag) and the Lyt-2/3 molecule during T cell activation was studied using the T cell clone KB5.C20, which is dependent upon Lyt-2 for target cell killing. This cytolytic T cell clone can be activated to secrete IFN-gamma by stimulation with H-2Kb expressing cells or with monoclonal antibodies directed against a clonotypic structure of the TcR or against associated CD3 molecules. IFN-gamma production induced by H-2Kb can be inhibited by anti-Lyt-2mAb. In addition, TcR-mediated activation using the anticlonotypic mAb Désiré-1 in soluble form can be inhibited by anti-Lyt-2 mAb in soluble form either as a divalent IgG or as its monovalent Fab fragment. Anti-Lyt-2 mAb immobilized on plastic wells was also inhibitory. Stimulation induced by the anti-TcR mAb or by anti-CD3 mAb immobilized on plastic can be inhibited only with plastic immobilized and not with soluble anti-Lyt-2mAb, however. These results are discussed in terms of local interactions between TcR and Lyt-2 molecules.