The induction of M phase in eukaryotic cell cycles requires robust activation of Cdc2/cyclin B by Cdc25, which itself is robustly activated by serine/threonine phosphorylations. Although multiple protein kinases that directly activate Cdc25C have been identified, whether the combination of different primary phosphorylations of Cdc25C is sufficient to fully activate Cdc25C has not been determined. By analyzing the GST-Cdc25C phosphorylating activity in Xenopus egg extracts, we previously defined roles of MAPK and Cdc2/cyclin B in partially activating Cdc25C and predicted the presence of another major Cdc25C-activating kinase. In this study, we demonstrate that this missing kinase is RSK2, which phosphorylates three sites in Cdc25C and also partially activates Cdc25C. However, the phosphorylations catalyzed by MAPK, Cdc2, and RSK2 fail to fully activate Cdc25C, suggesting that additional biochemical events are required to fully activate this key cell cycle regulator.