Listeria monocytogenes- (LM) antigen-dependent cytotoxic lymphocytes, identified in a companion manuscript as T cells (CTL), can kill a wide variety of target cells, including syngeneic fibroblasts of both fetal and adult origin, and certain allogeneic and xenogeneic tumor cells. Cold target cell inhibition assays revealed that cells susceptible to lysis can block the cytolytic process in a dose-dependent manner, whereas lysis-resistant cells cannot. Several lines of evidence indicate that to realize their cytolytic potential, LM-dependent CTL must bind to their targets. Aggressor cells that adhere to susceptible target cell monolayers have enhanced cytolytic activity when compared with unfractionated cells or cells that do not adhere. LM-dependent CTL fail to kill when they are separated from susceptible targets by an agar barrier. Arguments against the complicity of a soluble mediator in the killing process derive from the rapid (within 3 hr) expression of cytotoxicity in circumstances where cell contact can occur, and the finding that spent medium in which LM-dependent CTL are either activated or assayed is devoid of cytolytic activity. The specificity of LM-dependent CTL suggests that these effector T cells recognize an as yet unidentified array of antigens that are expressed on a variety of rodent cells. Their cytolytic repertoire cannot be ascribed solely to the polyclonal activation of alloreactive T cells, because responder cells that have been negatively selected for a particular RT-1 haplotype can kill target cells bearing these alloantigens.