We examined whether impairment of intracellular Ca² homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca² oscillations at fertilization: these Ca² oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca² store was decreased in in vitro-aged oocytes. A decrease in the Ca² store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca² -ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca² oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca² homeostasis (such as a decrease in the ER Ca² store) caused an alteration in Ca² oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H₂O₂ or thapsigargin. Acting together, alteration in Ca² oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.