Activation of mitogen-activated protein kinase (MAPK) in maturing mouse oocytes occurs after synthesis of Mos, a MAPKKK. To investigate whether Mos acts only through MEK1, we microinjected constitutively active forms of MEK1 (MEK1S218D/S222D referred herein as MEK*) and Raf (DeltaRaf) into mouse oocytes. In mos(-/-) oocytes, which do not activate MAPK during meiosis and do not arrest in metaphase II, MEK* and DeltaRaf did not rescue MAPK activation and metaphase II arrest, whereas Mos induced a complete rescue. MEK* and DeltaRaf induced cleavage arrest of two-cell blastomeres. They induced MAPK activation when protein phosphatases were inhibited by okadaic acid, suggesting that Mos may inhibit protein phosphatases. Finally, in mos(-/-) oocytes, MEK* induced the phosphorylation of Xp42(mapk)D324N, a mutant less sensitive to dephosphorylation, showing that a MAPK phosphatase activity is present in mouse oocytes. We demonstrate that active MAPKK or MAPKKK cannot substitute for Mos to activate MAPK in mouse oocytes. We also show that a phosphatase activity inactivates MAPK, and that Mos can overcome this inhibitory activity. Thus Mos activates MAPK through two opposite pathways: activation of MEK1 and inhibition of a phosphatase.