The basic principle of all proteolytic pathways is to achieve activation of an apical "initiator" protease, which can directly activate a downstream zymogen. In apoptosis, two distinct pathways -- an extrinsic pathway and an intrinsic one -- activate cysteine proteases in the caspase cascade. Intrinsic death pathway is triggered by signals that converge on the mitochondrion, activating another initiating caspase, caspase-9, which then activates the executioners. Alternative, the extrinsic death pathway conveys signals through ligation of TNF death receptor family members. Within the immune system, macrophages express both the canonical death receptor, Fas (CD95), a member of the TNF death receptor family, and its cognate ligand, FasL. Macrophages naturally do not die in response to Fas stimulation. However, macrophages with Fas have activated downstream "executioner" caspase-3 and caspase-3 substrates, such as PARP, are cleaved, but they do not die measured by TUNEL assay; while Fas-deficient macrophages do not have activated caspase-3. We propose to immunoprecipitate both pro- and activated-form of caspase-3 complexes from macrophages, with and without death, to be analyzed by combining high resolution 2D polyacrylamide gel electrophoresis, with mass spectrometry to identify binding partners to active caspase-3. In addition, using isotope coded affinity tags and tandem mass spectrometry, we can identify the ratios of the original caspase substrates from macrophages with and without functional Fas. Both methods together will provide insight into non-apoptotic functions of active caspases in cells.