Previous studies have demonstrated that phosphorylation of human p53 on serine 15 contributes to protein stabilization after DNA damage and that this is mediated by the ATM family of kinases. However, cellular exposure to hypoxia does not induce any detectable level of DNA lesions compared to ionizing radiation, and the oxygen dependency of p53 protein accumulation differs from that of HIF-1, the hypoxia-inducible transcription factor. Here we show that, under severe hypoxic conditions, p53 protein accumulates only in S phase and this accumulation correlates with replication arrest. Inhibition of ATR kinase activity substantially reduces hypoxia-induced phosphorylation of p53 protein on serine 15 as well as p53 protein accumulation. Thus, hypoxia-induced cell growth arrest is tightly linked to an ATR-signaling pathway that is required for p53 modification and accumulation. These studies indicate that the ATR kinase plays an important role during tumor development in responding to hypoxia-induced replication arrest, and hypoxic conditions could select for the loss of key components of ATR-dependent checkpoint controls.