Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors N-tosyl-L-lysyl chloromethylketone (TLCK) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK-sensitive target is pre-existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin-like protease which is a common effector of thymocyte apoptosis.