Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone (Z-VAD.FMK) inhibit apoptosis. N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) has a very different effect, inducing the early morphological and biochemical changes associated with apoptosis. TLCK inhibits trypsin-like proteases whilst Z-VAD.FMK inhibits interleukin-1 beta-converting enzyme (ICE)-like proteases; this and the contrasting effects of TPCK support the hypothesis that thymocyte apoptosis involves a hierarchy of proteases which act at different stages of the process.