Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, and Cdk2-cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism.