The role of T cell differentiation antigens in antigen-specific and nonspecific cytotoxicity by human cytotoxic T lymphocyte (CTL) clones was investigated. In contrast to other reports, several monoclonal antibodies (mAb) against the T3 antigen only marginally blocked antigen-specific cytotoxicity at high concentrations but induced cytotoxicity against third party cells at concentrations from 10 to 0.001 micrograms/ml. Susceptibility to anti-T3-induced lysis was variable but was found with all target cells. Incubation of CTL with anti-T3 mAb even led to self-destruction of the CTL. The effect was independent of the presence of Fc receptors on the target cell and could be obtained with F(ab')2 fragments of the antibody as well. Only activated but not resting T cells could be induced to lyse by anti-T3. Furthermore, this type of bystander killing of target cells could also be induced by the Ca2+ ionophore A23187. Antibodies against the T8 differentiation antigen inhibited antigen-specific, oxidation-induced and anti-T3-induced cytotoxicity by T8+ CTL clones, whereas triggering by the ionophore A23187 was not inhibited. These results show that undirected killing can be triggered in CTL by activating a transducing molecule directly without involving the antigen receptor. Since this triggering of the lethal hit can still be inhibited by mAb against the T8 molecule, the T8 molecule probably has a regulatory role in a late phase of CTL triggering.