Previous observations of ovulation and fertilization defects in cyclooxygenase-2 (COX-2)-deficient mice suggested that COX-2-derived ovarian prostaglandins (PGs) participate in these events. However, the specific PG and its mode of action were unknown. Subsequent studies revealed that mice deficient in EP₂, a PGE₂-receptor subtype, have reduced litter size, apparently resulting from poor ovulation but more dramatically from impaired fertilization. Using a superovulation regimen and in vitro culture system, we demonstrate herein that the ovulatory process, not follicular growth, oocyte maturation, or fertilization, is primarily affected in adult COX-2- or EP₂-deficient mice. Furthermore, our results show that in vitro-matured and -fertilized eggs are capable of subsequent preimplantation development. However, severely compromised ovulation in adult COX-2- or EP₂-deficient mice is not manifested in immature (3-wk-old) COX-2- or EP₂-deficient mice, suggesting that the process of ovulation is more dependent on PGs in adult mice. Although the processes of implantation and decidualization are defective in COX-2(−/−) mice, our present results demonstrate that these events are normal in EP₂-deficient mice, as determined by embryo transfer and experimentally induced decidualization. Collectively, previous and present results suggest that whereas COX-2-derived PGE₂ is essential for ovulation via activation of EP₂, COX-2-derived prostacyclin is involved in implantation and decidualization via activation of peroxisome proliferator-activated receptor δ.