Elevated level of oxygen (hyperoxia) is widely used in critical care units, and in respiratory insufficiencies. In addition, hyperoxia has been implicated in many diseases such as BPD or ARDS. Although hyperoxia is known to cause DNA base modifications and strand breaks the DNA damage response has not been adequately investigated. We have investigated the effect of hyperoxia on DNA damage signaling, and show that hyperoxia is a unique stress that activates ATR-dependent p53 phosphorylations (ser-6, -15, -37, 392), phosphorylation of histone H2AX (ser-139), and phosphorylation of Chk1. In addition, we show that phosphorylation of p53 (ser6) and histone H2AX (ser-139) depend on both ATR and ATM. We demonstrate that ATR activation precedes ATM activation in hyperoxia. Finally, we show that ATR is required for ATM activation in hyperoxia. Taken together, we report that ATR is the major DNA damage signal transducer in hyperoxia that activates ATM.