To understand the physiological significance of erythrocyte membrane aspartic proteinase (EMAP), its catalytic properties and possible mechanisms for its activation were investigated, and the results are as follows. (1) The enzyme was indistinguishable from cathepsin E but not cathepsin D enzymatically and immunochemically. (2) The enzyme was found at high levels in neutrophils, lymphocytes and platelets as well as erythrocytes. (3) The membrane-bound latent enzyme was activated by various procedures that induce the disturbance of normal erythrocyte membrane organization, e.g., by treatments with phospholipase C and trypsin or simply by heating at 40°C. The activation was accompanied by dissociation of the enzyme from the membrane. (4) When the young erythrocyte obtained by density gradient centrifugation on Percoll gradients were aged in vitro, a decrease in the membrane-bound form of EMAP and an increase in the soluble form were observed in a time-dependent manner. The erythrocyte aging also caused an increase in the membrane-bound form of cytosolic proteins such as hemoglobin.