Cytotoxic lymphocytes utilize granule associated serine proteases (granzymes) and perforin to induce apoptosis. Although the importance of granzyme B has been established by gene ablation experiments, biochemical events initiated by the granzyme remain enigmatic. We show here that exposure of Jurkat cells to granzyme B and perforin results in cleavage of poly(ADP-ribose) polymerase to an apoptotic 89 kDa fragment and to lesser amounts of a 64 kDa fragment. The 64 kDa fragment is produced directly by granzyme B while the 89 kDa fragment is presumably generated by activated ICE/Ced-3 proteases. Establishing the intracellular function of GrB in the apoptotic response, these results indicate that granzyme B enters perforin treated targets activating the ICE/Ced-3 family proteases which then cleave poly(ADP-ribose) polymerase to its apoptotic fragment. Intracellular granzyme B appears to be translocated to the nucleus where the protease directly cleaves poly(ADP-ribose) polymerase.