Two pathways have been implicated in the induction of apoptosis by cytotoxic T cells: the granule exocytosis pathway and a pathway using CD95 (Fas/APO-1). To test whether apoptosis induced by either of these pathways could be blocked by Bcl-2, we exposed bcl-2-transfected cells to CTL derived from normal, perforin-deficient, or CD95 ligand mutant (gld) mice. Although the levels of Bcl-2 expression achieved were able to protect FDC-P1 and Yac-1 transfectants from a variety of apoptotic stimuli, the cells were not protected from cytolysis mediated by CTL from any of these sources, by NK cells, or granules isolated from CTL. However, Bcl-2 expression significantly inhibited apoptosis induced by purified granzyme B and perforin. These results suggest that while Bcl-2 is capable of inhibiting the apoptotic pathway utilized by perforin and granzyme B, other granule components can bypass this block. We conclude that CTL harbor potent killing mechanism(s) in addition to those provided by CD95 ligand or perforin and granzyme B that cannot be overcome by Bcl-2.