Changes in ionic homeostasis are early events leading up to the commitment to apoptosis. While the direct effects of cations on caspase-3 activity have been examined, comparable studies on procaspase-3 are lacking. In addition, the effects of salts on caspase structure have not been examined. We have studied the effects of cations on the activities and conformations of caspase-3 and of an uncleavable mutant of procaspase-3 that is enzymatically active. The results show that caspase-3 is more sensitive to changes in pH and ion concentrations than is the zymogen. This is due both to the loss of an intact intersubunit linker and to the loss of the pro-domain. The results show that while the caspase-3 subunits reassemble to the heterotetramer, the activity return is low after the protein is incubated at or below pH 4.5 then returned to pH 7.5. The data further show that the irreversible step in assembly results from heterotetramer rather than heterodimer dissociation and demonstrate that the active site does not form properly following reassembly. However, active site formation is fully reversible when reassembly occurs in the presence of the pro-domain, and this effect is specific for the pro-peptide of caspase-3. The data show that the pro-domain facilitates both dimerization and active site formation in addition to stabilizing the native structure. Overall, the results show that the pro-domain acts as an intramolecular chaperone during assembly of the (pro)caspase subunits and increases the efficiency of formation of the native conformation.