Cumulus cell-oocyte complex (COC) expansion is obligatory for LH-induced ovulation and is initiated by LH induction of the epidermal growth factor (EGF)-like factors that mediate the synthesis of the hyaluronan-rich matrix and hyaluronan-stabilizing factors. COC expansion also involves the movement of cumulus cells within the matrix by mechanisms that have not been characterized. We document herein that two proteases, calpain 2 and to a lesser extent calpain 1, are expressed in cumulus cells and that the proteolytic activity of these enzymes is rapidly and significantly increased in COC isolated from human chorionic gonadotropin-induced ovulatory follicles in vivo. Stimulation of calpain activity was associated with proteolytic degradation of paxillin and talin (two components of focal adhesion complexes), cell detachment, and the formation of cell surface bleb-like protrusions. Injection of a calpain inhibitor in vivo reduced 1) human chorionic gonadotropin-stimulated calpain enzyme activity, 2) cell detachment, 3) membrane protrusion formation, and 4) COC expansion by mechanisms that did not alter Has2 expression. During EGF-like factor induction of COC expansion in culture, calpain activity was increased by ERK1/2 and intracellular Ca(2+) signaling pathways. Inhibition of calpain activity in cultured COC blocked cumulus cell detachment, protrusion formation, and the vigorous movement of cumulus cells. As a consequence, COC expansion was impaired. Collectively, these results show that two highly coordinated processes control COC expansion. One process involves the synthesis of the hyaluronan matrix, and the other mediates cumulus cell detachment and movement. The latter are controlled by calpain activation downstream of the EGF receptor activation of the Ca(2+) pathway and ERK1/2 pathways.