Beta-TrCP, the F-box protein of the SCF(beta-TrCP) ubiquitin ligase (SCF, Skp1/Cul1/F-box protein), recognizes the doubly phosphorylated DSG motif (DpSGPhiXpS) in various SCF(beta-TrCP) target proteins. The Cdc25A phosphatase, a key cell-cycle regulator in vertebrate cells, undergoes a rapid ubiquitin-dependent degradation in response to genotoxic stress. Beta-TrCP binds to the DSG motif of human Cdc25A in a manner dependent on Chk1 and other unknown kinases. However, Xenopus Cdc25A does not have a DSG motif at the corresponding site of human Cdc25A. Here, we report that both Xenopus Cdc25A and human Cdc25A have a previously undescribed nonphosphorylated DDG motif (DDGPhiXD) for recognition by beta-TrCP. When analyzed by using Xenopus eggs, the binding of beta-TrCP to the DDG motif is essential for the Chk1-induced ubiquitination and degradation of Xenopus Cdc25A and also plays a role in the degradation of human Cdc25A. The DDG motif also exists in human Cdc25B phosphatase (another key cell-cycle regulator), binds beta-TrCP strongly, and is essential for the ubiquitination and degradation of the (labile) phosphatase in normal conditions. We provide strong evidence that, in both Cdc25A and Cdc25B, the binding (efficiency) of beta-TrCP to the DDG motif is regulated by nearby residues, while ubiquitination is regulated by other events in addition to the beta-TrCP binding. Finally, our additional data suggest that beta-TrCP may recognize nonphosphorylated DDG-like motifs in many other proteins, including X11L (a putative suppressor of beta-amyloid production) and hnRNP-U (a pseudosubstrate of SCF(beta-TrCP)).