Cytolytic T lymphocytes kill target cells by two independent cytolytic mechanisms. One pathway depends on the polarized secretion of granule-stored proteins including perforin and granzymes, causing target cell death through membrane and DNA damage. The second cytolytic effector system relies on the interaction of the Fas ligand (Fasl) on the effector cell with its receptor (Fas) on the target cell, leading to apoptotic cell death. Using mixed lymphocyte culture (MLC)-derived primary T lymphocytes of perforin-knockout and gld (with non-functional FasL) mice, the molecular basis of the two killing mechanisms was compared. The activity of both pathways was dependent on extracellular Ca2+. Incubation of MLC-stimulated primary T cells with protein synthesis inhibitors prior to TCR triggering impaired FasL cell surface expression and abolished cytolytic activity, although the cells exhibited an intracellular pool of FasL. The perforin-dependent mechanism induced cell death more rapidly, although both pathways ultimately showed similar killing efficiencies. Both pathways induced comparable levels of DNA degradation, but Fas-induced membrane damage was less pronounced. We conclude that upon TCR triggering FasL may be recruited in part from pre-existing intracellular stores. However, efficient induction of target cell death still depends on the continuous biosynthesis of FasL molecules.