This study investigates the regulation of human Chk1 by ATR-mediated checkpoint pathways. Chk1 is a conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. The study demonstrates that agents that block DNA replication or cause DNA damage induce the phosphorylation of human Chk1. Phosphorylated Chk1 has higher intrinsic kinase activity and elutes more quickly on gel filtration columns. Serines 317 and 345 were identified as phosphorylation sites in vivo, and ATR phosphorylates these sites in vitro. Phosphorylation of Chk1 on these sites is ATR-dependent. Mutants of Chk1 with alanine substitutions at these sites are poorly activated in response to replication blocks or genotoxic stress and are poorly phosphorylated by ATR in vitro. These findings show that Chk1 activation in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. The study also implicates ATR as a direct upstream activator of Chk1 in human cells. Checkpoints are signaling pathways that monitor the integrity and replication status of genetic material before cells commit to DNA replication or segregation. Upon activation, checkpoints interface with cyclin-Cdk complexes to block cell cycle progression or induce cell death. The DNA replication checkpoint monitors S-phase completion and prevents mitosis in its absence. The DNA damage checkpoint monitors genome integrity and arrests the cell cycle in G1, S, or G2 phases. Eukaryotic cells activate a conserved set of checkpoint proteins to prevent replication or segregation of damaged DNA before repair. ATM is a key component of the DNA damage checkpoint, a 370-kDa protein kinase. ATM is mutated in ataxia telangiectasia and is involved in checkpoint responses to ionizing radiation. ATR, another kinase, also contributes to checkpoint control in eukaryotes. Unlike ATM, ATR is essential for embryonic development. ATR and ATM have overlapping functions in vivo, with similar kinase specificities. Both prefer phosphorylating serine or threonine residues followed by glutamine (SQ/TQ motifs). Chk1 is a component of signaling pathways that respond to DNA damage and incomplete DNA replication. In fission yeast, Chk1 responds to DNA damage induced by IR or UV and functions in the DNA replication checkpoint. In Xenopus, Chk1 responds to DNA replication blocks and UV damage but not to DNA with double-strand breaks. Chk1 is phosphorylated and activated by ATR in Xenopus, and the ATR-Chk1 pathway responds to unreplicated DNA and UV-damaged DNA. In human cells, Chk1 is phosphorylated on serines 317 and 345 in response to checkpoint activation. The phosphorylated, activated form of Chk1 elutes more quickly on gel filtration columns. ATR directly phosphorylatesThis study investigates the regulation of human Chk1 by ATR-mediated checkpoint pathways. Chk1 is a conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. The study demonstrates that agents that block DNA replication or cause DNA damage induce the phosphorylation of human Chk1. Phosphorylated Chk1 has higher intrinsic kinase activity and elutes more quickly on gel filtration columns. Serines 317 and 345 were identified as phosphorylation sites in vivo, and ATR phosphorylates these sites in vitro. Phosphorylation of Chk1 on these sites is ATR-dependent. Mutants of Chk1 with alanine substitutions at these sites are poorly activated in response to replication blocks or genotoxic stress and are poorly phosphorylated by ATR in vitro. These findings show that Chk1 activation in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. The study also implicates ATR as a direct upstream activator of Chk1 in human cells. Checkpoints are signaling pathways that monitor the integrity and replication status of genetic material before cells commit to DNA replication or segregation. Upon activation, checkpoints interface with cyclin-Cdk complexes to block cell cycle progression or induce cell death. The DNA replication checkpoint monitors S-phase completion and prevents mitosis in its absence. The DNA damage checkpoint monitors genome integrity and arrests the cell cycle in G1, S, or G2 phases. Eukaryotic cells activate a conserved set of checkpoint proteins to prevent replication or segregation of damaged DNA before repair. ATM is a key component of the DNA damage checkpoint, a 370-kDa protein kinase. ATM is mutated in ataxia telangiectasia and is involved in checkpoint responses to ionizing radiation. ATR, another kinase, also contributes to checkpoint control in eukaryotes. Unlike ATM, ATR is essential for embryonic development. ATR and ATM have overlapping functions in vivo, with similar kinase specificities. Both prefer phosphorylating serine or threonine residues followed by glutamine (SQ/TQ motifs). Chk1 is a component of signaling pathways that respond to DNA damage and incomplete DNA replication. In fission yeast, Chk1 responds to DNA damage induced by IR or UV and functions in the DNA replication checkpoint. In Xenopus, Chk1 responds to DNA replication blocks and UV damage but not to DNA with double-strand breaks. Chk1 is phosphorylated and activated by ATR in Xenopus, and the ATR-Chk1 pathway responds to unreplicated DNA and UV-damaged DNA. In human cells, Chk1 is phosphorylated on serines 317 and 345 in response to checkpoint activation. The phosphorylated, activated form of Chk1 elutes more quickly on gel filtration columns. ATR directly phosphorylates