Although considerable information about calpain has been obtained from studies using isolated enzyme preparations, this does not permit the dynamic assessment of intracellular protease activity during changes in intracellular Ca2+ and pH. Intracellular calpain activity is regulated by complex interactions with the specific inhibitory protein (calpastatin), Ca2+ and phospholipids, conditions which are difficult to duplicate in isolated systems. Thus a method to measure calpain activity over time in living cells would be helpful in studying its regulation and physiological roles.