Chk2/hCds1, a human homolog of the yeast RAD53/SPK1 and fission yeast cdc1 checkpoint genes, functions as a DNA damage checkpoint in G1 by stabilizing the p53 tumor suppressor protein. In response to DNA damage, Chk2/hCds1 phosphorylates p53 on Ser-20, disrupting its interaction with Mdm2, a protein that targets p53 for degradation. This phosphorylation is ATM-dependent. In human cells, Chk2/hCds1-induced p53 stabilization leads to G1 arrest, which is inhibited by a dominant-negative p53 mutant. Ectopic expression of wild-type Chk2/hCds1 enhances p53 stabilization and G1 arrest, suggesting that Chk2/hCds1 is a key component of the DNA damage checkpoint. The findings suggest a revised model for the checkpoint-signaling pathways leading to p53 stabilization, where Chk2/hCds1 acts as an intermediary between DNA damage and p53 regulation.Chk2/hCds1, a human homolog of the yeast RAD53/SPK1 and fission yeast cdc1 checkpoint genes, functions as a DNA damage checkpoint in G1 by stabilizing the p53 tumor suppressor protein. In response to DNA damage, Chk2/hCds1 phosphorylates p53 on Ser-20, disrupting its interaction with Mdm2, a protein that targets p53 for degradation. This phosphorylation is ATM-dependent. In human cells, Chk2/hCds1-induced p53 stabilization leads to G1 arrest, which is inhibited by a dominant-negative p53 mutant. Ectopic expression of wild-type Chk2/hCds1 enhances p53 stabilization and G1 arrest, suggesting that Chk2/hCds1 is a key component of the DNA damage checkpoint. The findings suggest a revised model for the checkpoint-signaling pathways leading to p53 stabilization, where Chk2/hCds1 acts as an intermediary between DNA damage and p53 regulation.