Cell-cycle progression is regulated by cyclin-dependent kinases (CDKs). CDK1 and CDK2 can be also activated by noncyclin proteins named RINGO/Speedy, which were identified as inducers of the G2/M transition in Xenopus oocytes. However, it is unclear how XRINGO triggers M phase entry in oocytes. We show here that XRINGO-activated CDKs can phosphorylate specific residues in the regulatory domain of Myt1, a Wee1 family kinase that plays a key role in the G2 arrest of oocytes. We have identified three Ser that are major phosphoacceptor sites for CDK/XRINGO but are poorly phosphorylated by CDK/cyclin. Phosphorylation of these Ser inhibits Myt1 activity, whereas their mutation makes Myt1 resistant to inhibition by CDK/XRINGO. Our results demonstrate that XRINGO-activated CDKs have different substrate specificity than the CDK/cyclin complexes. We also describe a mechanism of Myt1 regulation based on site-specific phosphorylation, which is likely to mediate the induction of G2/M transition in oocytes by XRINGO.