Induction of T-cell activation requires multiple signals provided by cell surface receptor interactions and/or cytokines. T-cell stimulation via the T-cell receptor/CD3 complex provides an important initial activation event which, when combined with the proper costimulatory signals, results in an activated effector T cell. In this report, we have investigated the effectiveness of epithelial glycoprotein-2- (EGP-2) positive tumor target cells to induce specific T-cell stimulation via CD3, CD5, and CD28 using various combinations of bispecific monoclonal antibodies (BsMab) directed against CD3, CD5, or CD28 on the one hand and the pancarcinoma-associated antigen EGP-2 on the other. Induction of T-cell activation was investigated by assessment of CD69 expression, induction of proliferation, and acquirement of cytolytic potential. EGP-2-specific induction of T-cell activation was observed using combinations of BsMab which simultaneous ligated CD3/CD5, CD3/CD28, or CD3/CD5/CD28 with EGP-2. Activation with CD3-, CD5-, or CD28-based BsMab alone did not result in significant induction of T-cell activation in the presence or absence of EGP-2-positive target cells. Simultaneous ligation via CD5/CD28 resulted in partial T-cell activation, including CD69 up-regulation and increased cytolytic activity. Stimulation via CD3 and CD5 or CD28 could be further increased by the addition of exogenously added recombinant Interleukin 2. In contrast, T-cell activation by simultaneous ligation of CD3/CD5/CD28 could not be further augmented by addition of exogenous interleukin 2, indicating that T-cell activation via the combination of CD3, CD5, and CD28 results in complete T-cell activation. Our results show that rapid and target cell-specific induction of T cells is possible using combinations of BsMab directed against different costimulatory molecules. Simultaneous costimulation via CD3/CD5/CD28 results in the most complete activation of T cells.