The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.