Phosphorylation at Ser-15 may be a critical event in the up-regulation and functional activation of p53 during cellular stress. In this report we provide evidence that the ATM–Rad3-related protein ATR regulates phosphorylation of Ser-15 in DNA-damaged cells. Overexpression of catalytically inactive ATR (ATR^ki) in human fibroblasts inhibited Ser-15 phosphorylation in response to γ-irradiation and UV light. In γ-irradiated cells, ATR^kiexpression selectively interfered with late-phase Ser-15 phosphorylation, whereas ATR^ki blocked UV-induced Ser-15 phosphorylation in a time-independent manner. ATR phosphorylated p53 at Ser-15 and Ser-37 in vitro, suggesting that p53 is a target for phosphorylation by ATR in DNA-damaged cells.