Cytochrome c is absolutely required in vitro system for the activation of caspase-3 and caspase-9. When cytochrome c was depleted from HeLa cell S-100 extracts by a monoclonal antibody, the ability to activate caspases was abolished4. t. When cytochrome c was depleted, caspase-9 failed to bind Apaf-1 even in the presence of dATP. The binding was restored when purified cytochrome c was added. These suggest that cytochrome c initiates apoptosis by inducing the formation of the Apaf-1/caspase-9 complex4.