Previously we have demonstrated that 150 mM KCl and alkaline pH each inhibits cytochrome c mediated activation of procaspase-3 in a unique manner. To determine the mechanism of inhibition we analyzed the effect that KCl and alkaline pH has on the formation of apoptosomes (a large complex consisting of cytochrome c, Apaf-1 and procaspase-9/caspase-9) in vitro. Our results suggest that an initial 700 kDa apoptosome matures through a 1.4 MDa intermediate before a 700 kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrated that 150 mM KCl interferes with the conversion of the initial 700 kDa apoptosome to the 1.4 MDa intermediate, while alkaline pH "traps" the apoptosome in the 1.4 MDa intermediate. Analysis of the cleaved state of procaspase-9 and procaspase-3 suggest that the 1.4 MDa intermediate may be required for cleavage of procaspase-9. Consistent with these results, in vivo data suggests that blocking acidification during induction of apoptosis inhibits activation of procaspase-3. Based on these results we propose a model of apoptosome maturation.