Apoptosis, or programmed cell death, is a fundamental biological process involved in many physiological and pathological phenomena. This process is predominantly catabolic in which cellular macromolecules are broken down by distinct enzymes to be later recycled in healthy cells. These enzymes are arranged in an elaborate cascade that serves to both propagate and amplify a death signal as well as process bulk macromolecules and inhibit repair systems. One of the best-characterized enzyme systems involved in apoptosis is the activation of a nuclease(s) that degrades the genome into discrete oligonucleosomal fragments, clearly committing a cell to death. Using two different in vitro models, we have shown that K+ directly inhibits this/these nuclease(s) with complete inhibition observed at cellular concentrations of K+ found in non-dying cells (150 mM). These data suggest that K+ concentrations in living cells suppress apoptotic nuclease activity. One of the nucleases involved in apoptotic DNA degradation (NUC18/Cyclophilin) has been purified in our laboratory and found to be inhibited by similar concentrations of K+. Upstream of DNA fragmentation a specific class of proteases, termed caspases, are activated which propagate an apoptotic signal and lead to downstream events such as DNA fragmentation. Using an in vitro model of caspase activation, we also observed that activation of this enzyme is also completely inhibited by normal physiological K+ levels, suggesting that K+ levels in non-dying cells suppress multiple portions of the apoptotic enzyme cascade. These results suggest that K+ concentrations may decrease in cells undergoing apoptosis and both physical and fluorescence techniques document an intracellular K+ concentration of 35 mM in apoptotic cells. This loss of K+ also accounts for changes in cell volume (cell shrinkage) that are universally associated with apoptosis. Measurements of K+ concentration in shrunken and non-shrunken cells demonstrated that only shrunken cells contain a reduced concentration of intracellular K+. Importantly, both the nuclease and caspase activity were found exclusively in this shrunken population providing an absolute correlation between the activity of these enzymes and low K+ levels within a cell. Finally, suppressing K+ efflux in whole cells prevents the activation of these enzymes whereas enhancing the efflux of this ion facilitates enzymatic activity. These results suggest a direct cause and effect relationship between the level of K+ and the activity of apoptotic enzymes. Taken together we have shown a critical and novel mechanism that regulates apoptosis by a direct effect on the apoptotic enzymes.