Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP–dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1–procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c and dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.