In a yeast 2-hybrid screen the activating transcription factor-4 (ATF-4) was identified as novel interactor of the oxygen sensor prolyl-4-hydroxylase domain (PHD)3. Using different deletion variants the zipperII domain (aa 89–123) of ATF-4 was identified as interaction domain. The protein interaction between ATF-4 and PHDs was verified in in vitro pull-down assays using MBP-PHDs and V5-tagged ATF-4. Previously it was described that ATF-4 is translationally induced under anoxic conditions by phosphorylation of eIF2a. Exposing HeLa cells to the PHD inhibitors DMOG, CoCl2 or to hypoxia, however, also stabilized ATF-4 in an eIF2a-independent manner. Oxygen-dependent expression was mediated by the amino acid stretch 154 to 183 adjacent to the zipper II. Mutation of all five prolines in this sequence resulted in stabilized ATF-4 in normoxia, demonstrating that oxygen-dependent expression is mediated by those prolines. Treatment of HeLa cells with siRNA targeting PHD2 or PHD3 resulted in increased expression of ATF-4 after downregulation of PHD3 but not PHD2. These data demonstrate that ATF-4 expression is mediated by PHD3.