MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1/caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ~1.4-MDa and ~700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ~700-kDa apoptosome complex which is also formed in apoptotic MCF-7 cells is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable ~200-kDa XIAP/caspase-7 complex, which does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3 containing cells, XIAP appears to have a more significant anti-apoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable ~200-kDa complex with active caspase-7.