m-Calpain, which usually requires near-millimolar Ca2+ for activation, undergoes autolysis at 25 microM Ca2+ in the presence of mu-calpain. m-Calpain in itself exhibits no sign of autolysis around this Ca2+ concentration. Half-maximal rate of the reaction occurs at 30 microM Ca2+, showing that it is mu-calpain that catalyzes the limited proteolysis of m-calpain in an intermolecular reaction ("heterolysis"). This heterolytic step is accompanied by the activation of m-calpain: mu- and m-calpain preincubated together at 25 microM Ca2+ show significantly higher activity than the sum of activities of mu- and m-calpains preincubated separately. m-Calpain is sensitized to Ca2+ by mu-calpain-mediated activation: the half-maximal value of 160 microM for activation is lowered to 64 microM, which is similar to the shift found in m-calpain autoactivation. We suggest that these in vitro observations are relevant in vivo and the calpain cascade may play an important role in coordinating the functioning of calpains in living cells.