ATR regulates p53 phosphorylation in response to DNA damage. This study shows that ATR, a member of the phosphoinositide-3 kinase-related kinase (PIK) family, is involved in the phosphorylation of p53 at Ser-15 and Ser-37 in DNA-damaged cells. Overexpression of a catalytically inactive ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to γ-irradiation and UV light. ATR phosphorylates p53 in vitro, suggesting that ATR directly modifies p53 in DNA-damaged cells. These findings indicate that ATR functions as an upstream regulator of p53 phosphorylation in DNA-damaged cells. ATR and ATM play overlapping and independent roles in the phosphorylation of p53 during cellular exposure to genotoxic stress. ATR is structurally related to ATM and is involved in DNA damage responses. The results suggest that ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in checkpoint pathways induced by DNA replication blocks, DNA strand breaks, and other chromosomal abnormalities. ATRki overexpression inhibits Ser-15 phosphorylation in γ-irradiated cells but not in UV-irradiated cells, indicating that ATR plays a role in the late-phase phosphorylation of p53 in response to γ-radiation. ATR also phosphorylates p53 at Ser-37. These findings suggest that ATR and ATM both contribute to the phosphorylation of p53 in response to DNA damage. ATR is involved in the DNA damage response and may function in parallel with ATM. ATR is involved in the activation of cell-cycle checkpoints induced by DNA damage. ATR and ATM are involved in DNA damage responses. ATR is involved in the phosphorylation of p53 at Ser-15 and Ser-37. ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in the phosphorylation of p53 in response to DNA damage. ATR is involved in the DNA damage response and may function in parallel with ATM. ATR is involved in the activation of cell-cycle checkpoints induced by DNA damage. ATR and ATM are involved in DNA damage responses. ATR is involved in the phosphorylation of p53 at Ser-15 and Ser-37. ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in the DNA damage response and may function in parallel with ATM. ATR is involved in the activation of cell-cycle checkpoints induced by DNA damage. ATR and ATM are involved in DNA damage responses. ATR is involved in the phosphorylation of p53 at Ser-15 and Ser-37. ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in theATR regulates p53 phosphorylation in response to DNA damage. This study shows that ATR, a member of the phosphoinositide-3 kinase-related kinase (PIK) family, is involved in the phosphorylation of p53 at Ser-15 and Ser-37 in DNA-damaged cells. Overexpression of a catalytically inactive ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to γ-irradiation and UV light. ATR phosphorylates p53 in vitro, suggesting that ATR directly modifies p53 in DNA-damaged cells. These findings indicate that ATR functions as an upstream regulator of p53 phosphorylation in DNA-damaged cells. ATR and ATM play overlapping and independent roles in the phosphorylation of p53 during cellular exposure to genotoxic stress. ATR is structurally related to ATM and is involved in DNA damage responses. The results suggest that ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in checkpoint pathways induced by DNA replication blocks, DNA strand breaks, and other chromosomal abnormalities. ATRki overexpression inhibits Ser-15 phosphorylation in γ-irradiated cells but not in UV-irradiated cells, indicating that ATR plays a role in the late-phase phosphorylation of p53 in response to γ-radiation. ATR also phosphorylates p53 at Ser-37. These findings suggest that ATR and ATM both contribute to the phosphorylation of p53 in response to DNA damage. ATR is involved in the DNA damage response and may function in parallel with ATM. ATR is involved in the activation of cell-cycle checkpoints induced by DNA damage. ATR and ATM are involved in DNA damage responses. ATR is involved in the phosphorylation of p53 at Ser-15 and Ser-37. ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in the phosphorylation of p53 in response to DNA damage. ATR is involved in the DNA damage response and may function in parallel with ATM. ATR is involved in the activation of cell-cycle checkpoints induced by DNA damage. ATR and ATM are involved in DNA damage responses. ATR is involved in the phosphorylation of p53 at Ser-15 and Ser-37. ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in the DNA damage response and may function in parallel with ATM. ATR is involved in the activation of cell-cycle checkpoints induced by DNA damage. ATR and ATM are involved in DNA damage responses. ATR is involved in the phosphorylation of p53 at Ser-15 and Ser-37. ATR and ATM have partially overlapping functions in the DNA damage response. ATR is involved in the