CTLs lyse Fas-expressing target cells by the concomitant action of a perforin- and a Fas-dependent mechanism. This study analyzed whether target cells pulsed with T cell antagonists and other altered peptide ligands (APLs) were susceptible selectively to only one of these two mechanisms. In vivo and in vitro activated T cells from transgenic mice expressing a TCR specific for lymphocytic choriomeningitis virus were used as effector cells. To distinguish between perforin- and Fas-dependent cytotoxicity, T cells from normal or perforin-deficient mice were used to lyse peptide-pulsed Fas-positive or Fas-negative target cells. In contrast to previous reports that have shown that APLs selectively induce the Fas-dependent pathway of cytotoxicity, our results demonstrate that target cells pulsed with T cell antagonists and other APLs are lysed predominantly by the perforin-dependent pathway. The contribution of Fas-mediated cytotoxicity was similar for the full agonist and the APLs. Thus, full agonists, partial agonists, and antagonists trigger similar and not distinct pathways of cytotoxicity.