Intrinsic pathway of Apoptosis is initiated by the release of cytochrome C from mitochondria leading to the formation of a mega Dalton complex called apoptosome. Apoptosome, which consists of seven molecules of Apaf-1, serves as a platform for the oligomerization and activation of procaspase-9. Procaspase-9 is autoprocessed to caspase-9 in order to activate the executioner caspases. The exact number of caspase-9 molecules that can associate with apoptosome is a matter of controversy. In addition, it is not known if the processing of procaspase-9 to caspase-9 alters the stability of apoptosome. This is important because the duration of apoptosome signaling is dependent upon this factor. Consequently, we have investigated the apoptosome with respect to stoichiometry of caspase-9 and apaf-1 proteins, the effect of ionic strength on apoptosome activity, and the stability of the complex along with its components. The optimum ratio of caspase-9 molecules per apoptosome complex was determined as 3.3, although caspase-9 activity was increased up to one to one ratio of caspase-9 to apaf-1. Increase in ionic strength resulted in reduced caspase-9 activity perhaps due to interference of salt with electrostatic interactions of the enzyme with the substrate. Thermal inactivation kinetics experiments showed that both Apaf-1 and caspase-9 are more stable than the complex of the two. It further showed that the inactivation of caspase-9 is a unimolecular process due to irreversible loss of the conformation while the inactivation of apaf-1 is due to intermolecular interactions. Interestingly, apoptosome in the presence of procaspase-9 was observed to be more stable than in the presence of caspase-9. Based on this study we hypothesize that the cleavage of procaspase-9 occurs to destabilize the apoptosome complex in order to limit the duration of the death signal.