Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2 and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 (Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 (Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate Ser37 in either ATM+/+ or ATM-/- cells. Furthermore, H2O2 failed to phosphorylate Ser15 in ATM-/- cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM+/+ as well as in ATM-/- cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2 or UV light.