Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA .In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis [2-5]. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2 [6-11]. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2 . Here we propose that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. We show by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. We conclude that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA.