BINDING of Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas-bearing cells(1). The interleukin-1 beta-converting enzyme (ICE) is a cysteine protease(2) that is involved in apoptosis induced by various stimuli, including Fas-mediated apoptosis(3-8). Several ICE homologues have been identified, and these are subdivided into three groups (ICE-, CPP32- and Ich-1-like proteases)(9-18). We show here that specific inhibitors of ICE- or CPP32-like proteases can inhibit Fas-mediated apoptosis. Transient ICE-like activity was found in the cytosolic fraction of Fas-activated cells, whereas ICE-dependent, CPP32-like activity gradually accumulated in the cytosol. Cell lysates from mouse lymphoma supplemented with either recombinant ICE or CPP32 induced apoptosis of nuclei. The CPP32 inhibitor inhibited ICE- or CPP32-induced apoptosis in the cell-free system, whereas the ICE-inhibitor only inhibited ICE-induced apoptosis. Cell extracts from thymocytes from ICE-null mice induced apoptosis in the cell-free system when it was supplemented with CPP32. These results indicate that Fas sequentially activates ICE- and CPP32-like proteases, and that downstream CPP32, together with a component(s) in the cytoplasm, causes apoptosis of nuclei.