Fertilizable mammalian oocytes are arrested at the second meiotic metaphase ( mII) by the cyclinB- Cdc2 heterodimer, maturation promoting factor ( MPF). MPF is stabilized via the activity of an unidentified cytostatic factor ( CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/ Emi2, which we term endogenous meiotic inhibitor 2 ( Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm- induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.