Cytolytic T lymphocytes (CTLs) are an efficient immune mechanism for the destruction of foreign or pathogenic cells. Attempts to use CTLs in human cancer therapy have focused on the cell-surface molecules that regulate CTL function. An important molecule in CTL function is the CD3 antigen. Biochemical characterization has suggested that the CD3 antigen may function as a "trigger" for T-lymphocyte activation. To investigate this possibility, we used monoclonal antibody (MAb) to the CD3 antigen to trigger activation of long-term CTL lines. The anti-CD3 MAb was able to trigger killing of a variety of human and mouse tumor cell lines; however, not all tumor cells were lysed by the CTL. The susceptibility of the tumor cells to CTL-mediated lysis appeared to correlate with the binding of the anti-CD3 MAb to the tumor cell surface. The requirement for surface binding of the MAb was tested by covalently cross-linking the anti-CD3 MAb to the tumor cell membrane. Membrane-bound anti-CD3 MAb triggered high levels of CTL-mediated tumor cell killing. Similar results were obtained when anti-CD3 MAb was cross-linked to phosphatidylethanolamine and inserted into the cell membrane. These results indicate that the attachment of anti-CD3 MAb to the tumor cell surface provides a powerful new approach to the in vitro activation of human killer T cells and the in vivo treatment of human cancer.