Cell death pathways and their regulation during development of the preimplantation embryo are poorly understood. Our previous studies indicate that Ca2+ is a key mediator of apoptosis in different cell models. We hypothesized that Ca2+ signaling mediates apoptotic cell death during early embryonic development via activation of Ca2+-dependent proteases, micro-calpain, and caspase-12. To test this hypothesis, established procedures for in vitro production of bovine embryos in concert with fluorescence, high-resolution, digital Ca2+ imaging, detection of Ca2+-dependent apoptotic mediators, and measurement of apoptotic cell death were used in the present studies. We found that an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the individual embryo cells (2.6-fold) and mature oocytes (1.6-fold) was associated with activation of micro-calpain, induction of pan-caspase activity (5-10-fold), and expression of the Ca2+-dependent caspase-12. Inhibition of calpain or caspase activity significantly (1.5-2-fold) reduced apoptotic indices in embryos treated with the mobilizer of intracellular Ca2+ stores, thapsigargin, or the Ca2+ ionophore, ionomycin. Taken together, these results support our hypothesis that Ca2+ is involved in apoptosis of the preimplantation bovine embryo and that Ca2+-dependent apoptotic proteases are micro-calpain and caspase-12.