The natural arrest of vertebrate unfertilized eggs in second meiotic metaphase results from the activity of cytostatic factor (CSF). The product of the c-mos(xe) proto-oncogene is thought to be a component of CSF and can induce metaphase arrest when injected into blastomeres of two-cell embryos. The c-Mos(xe) protein can directly activate the mitogen-activated protein kinase kinase (MAP kinase kinase) in vitro, leading to activation of MAP kinase. MAP kinase and c-Mos(xe) are active in unfertilized eggs and are rapidly inactivated after fertilization. Microinjection of thiophosphorylated MAP kinase into one blastomere of a two-cell embryo induced metaphase arrest similar to that induced by c-Mos(xe). However, only arrest with c-Mos(xe) was associated with activation of endogenous MAP kinase. These results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of C-Mos(xe) is mediated by MAP kinase.