This study investigates the role of the ATM-Rad3-related protein ATR in the phosphorylation of p53 at Ser-15 in response to DNA damage. Overexpression of a catalytically inactive form of ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to γ-irradiation and UV light. ATRki specifically interfered with late-phase Ser-15 phosphorylation after γ-irradiation but blocked UV-induced Ser-15 phosphorylation in a time-independent manner. In vitro, ATR phosphorylated p53 at Ser-15 and Ser-37, suggesting that ATR is a direct regulator of p53 phosphorylation in DNA-damaged cells. The results indicate that ATR and ATM play both overlapping and independent roles in the phosphorylation of p53 during cellular exposure to genotoxic stress. ATR is proposed to function as an upstream regulator of p53 phosphorylation, with ATM and ATR potentially functioning sequentially in the response to DNA damage.This study investigates the role of the ATM-Rad3-related protein ATR in the phosphorylation of p53 at Ser-15 in response to DNA damage. Overexpression of a catalytically inactive form of ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to γ-irradiation and UV light. ATRki specifically interfered with late-phase Ser-15 phosphorylation after γ-irradiation but blocked UV-induced Ser-15 phosphorylation in a time-independent manner. In vitro, ATR phosphorylated p53 at Ser-15 and Ser-37, suggesting that ATR is a direct regulator of p53 phosphorylation in DNA-damaged cells. The results indicate that ATR and ATM play both overlapping and independent roles in the phosphorylation of p53 during cellular exposure to genotoxic stress. ATR is proposed to function as an upstream regulator of p53 phosphorylation, with ATM and ATR potentially functioning sequentially in the response to DNA damage.