Autocatalytic activation of initiator caspases is the link between pro-apoptotic signals and the destruction machinery of apoptosis. Activation of caspase-9, which mediates oncogene and drug-induced apoptosis, requires binding to the protein APAF-1. We found that the proteolytic activity of caspase-9 in a complex with APAF-1 is several orders of magnitude higher than that of the free enzyme. Thus, this complex functions as a holoenzyme in which caspase-9 is the catalytic subunit and APAF-1 its allosteric regulator. We argue that caspase-9 is activated by allosteric regulation and suggest that this mechanism is common for other initiator caspases.