Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2␣ at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2␣ was able to attenuate the phosphorylation of eIF2␣ and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2␣-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.Mammalian cells have the ability to alter their gene expression in order to adapt to a variety of environmental stresses, including nutrient depletion, oxidative stress, UV irradiation, reductive stress, exposure to toxins, and hypoxia, although the exact molecular events controlling the stress response have not been fully elucidated.Hypoxic stress plays a pivotal role in normal human development and physiology, including embryogenesis and wound repair, and has been well studied for its importance in the pathogenesis of several human diseases, namely, heart disease, stroke, diabetes, and cancer (for a detailed review, see reference 52). Hypoxia results when oxygen availability does not meet the demand of the surrounding tissue, resulting in decreased oxygen tension. In cancer this is initiated by the rapid proliferation of tumor cells, which gives rise to abnormal and chaotic vasculature, leading to the development of occlusions, blind ends, and vesicular shunts (4). The presence of hypoxic cells in solid tumors is well documented, and both clinical and experimental evidence suggests that the hypoxic microenvironment of a tumor helps to produce a more aggressive phenotype (62) by functioning as a selective pressure for the clonal expansion of apoptotically insensitive cells (16). The presence of hypoxic regions in solid tumors also correlates with a poor prognosis and has been shown to limit the efficacy of standard anticancer treatments, such as radiotherapy and chemotherapy (5). A key element to cellular survival and adaptation during hypo...