This study investigates the regulation of human Chk1, a protein kinase involved in cell cycle progression in response to checkpoint activation. The authors found that agents blocking DNA replication or causing DNA damage induce the phosphorylation of human Chk1 on serines 317 and 345. Phosphorylated Chk1 exhibits higher intrinsic kinase activity and elutes more quickly on gel filtration columns. Serines 317 and 345 were identified as the sites of phosphorylation in vivo, and ATR (ATM- and Rad3-related protein kinase) was shown to phosphorylate these sites in vitro. The phosphorylation of Chk1 on these sites was found to be dependent on ATR. Mutants of Chk1 with alanine substitutions at these sites were less activated in response to replication blocks or genotoxic stress, were poorly phosphorylated by ATR, and did not elute as quickly on gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345, and implicate ATR as a direct upstream activator of Chk1 in human cells.This study investigates the regulation of human Chk1, a protein kinase involved in cell cycle progression in response to checkpoint activation. The authors found that agents blocking DNA replication or causing DNA damage induce the phosphorylation of human Chk1 on serines 317 and 345. Phosphorylated Chk1 exhibits higher intrinsic kinase activity and elutes more quickly on gel filtration columns. Serines 317 and 345 were identified as the sites of phosphorylation in vivo, and ATR (ATM- and Rad3-related protein kinase) was shown to phosphorylate these sites in vitro. The phosphorylation of Chk1 on these sites was found to be dependent on ATR. Mutants of Chk1 with alanine substitutions at these sites were less activated in response to replication blocks or genotoxic stress, were poorly phosphorylated by ATR, and did not elute as quickly on gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345, and implicate ATR as a direct upstream activator of Chk1 in human cells.