The Caenorhabditis elegans cell-death gene ced-3 encodes a protein similar to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in mammalian apoptosis. We show that the full-length CED-3 protein undergoes proteolytic activation to generate a CED-3 cysteine protease and that CED-3 protease activity is required for killing cells by programmed cell death in C. elegans. We developed an easy and general method for the purification of CED-3/ICE-like proteases and used this method to facilitate a comparison of the substrate specificities of four different purified cysteine proteases. We found that in its substrate preferences CED-3 was more similar to the mammalian CPP32 protease than to mammalian ICE or NEDD2/ICH-1 protease. Our results suggest that different mammalian CED-3/ICE-like proteases may have distinct roles in mammalian apoptosis and that CPP32 is a candidate for being a mammalian functional equivalent of CED-3.