The tumour suppressor activity of p53 in vivo can be subject to pressure from the physiological stress of hypoxia and we report on the development of a cell system to define the p53-dependent stages in the adaptation of cells to hypoxia. p53+/+ cells exposed to hypoxia exhibited a transient arrest in G2/M, but escaped from this checkpoint and entered a long-term G 0/G 1 arrest. By contrast, isogenic p53-null cells exposed to hypoxic conditions exhibited a 6–10-fold higher level of apoptosis, suggesting that p53 acts as a survival factor under limiting oxygen concentrations. Surprisingly, hypoxia-dependent growth arrest in p53+/+ cells did not result in either p21 WAF1 or HIF-1 protein stabilization, but rather promoted a significant decrease in Ser 392-site phosphorylation at the CK2/FACT site. However, chemically induced anoxia induced Ser 392-site phosphorylation as well as stabilization of both p53 and HIF-1 proteins. In contrast to hypoxia, 5-flourouracil (5-FU)-induced p53-dependent cell death correlated with enhanced Ser 392 phosphorylation of p53 and elevated p21 WAF1 protein levels. Hypoxia inhibited 5-FU-induced p53-dependent cell death and attenuated p53 phosphorylation at the ATM and CK2/FACT phosphorylation sites. Although anoxia activates the p53 response, hypoxia silences the p53 transactivation pathway and identifies a physiological signalling model to study mechanisms of p53 inactivation under hypoxic conditions.