Human cloned cytotoxic T lymphocytes (CTL) specific for class I HLA antigens were used to investigate whether triggering of CTL leads to killing of innocent bystander target cells. After triggering of CTL by their specific target cells, lysis of bystander targets was detected in a 7-h cytotoxicity assay. Considerable differences were found in the susceptibility of various target cells to this type of lysis. Targets susceptible to this bystander lysis were also susceptible to lysis by CTL triggered by F(ab')2 fragments of an anti-T3 monoclonal antibody, whereas other targets were resistant to both types of cytotoxicity. Triggering of CTL by oxidized target cells or via a T3-independent activation pathway led to bystander lysis detectable already after 4 h. Bystander lysis was considerably enhanced under conditions that facilitated a non-specific cell contact between CTL and bystander target. We conclude that a function besides antigen recognition of the T cell receptor on CTL is to direct killing to the target cell. This directing, however, is incomplete and destruction of innocent bystanders can be detected under appropriate conditions.