Objective: Hypoxia-inducible factors (HIFs) are transcription factors involved in adaptation to reduced oxygen availability. In normoxia prolyl hydroxylase domain (PHD) proteins hydroxylate the HIF- subunits for subsequent polyubiquitination by von Hippel-Lindau protein and proteasomal degradation. As reported earlier PHD1 is present exclusively in the nucleus, PHD2 is mainly cytoplasmic and PHD3 is located both in the nucleus and the cytoplasm. The purpose of this study was to investigate the molecular mechanisms regulating intracellular allocation of PHD2. Methods: By inhibition of CRM1-dependent nuclear export, nuclear-cytoplasmic trafficking of PHD2 was analyzed. Co-immunoprecipitation and glutathione-S-transferase (GST) pulldown experiments were performed in order to detect protein-protein interactions between PHD2 and several nuclear transport receptors. Nuclear localization signal (NLS) and nuclear export signal (NES) mutants of PHD2 were generated and primary structure elements essential for PHD2 localization were mapped. Moreover, we investigated the impact of differences in the intracellular localization of PHD2 on HIF-1a transcriptional activity by reporter gene assays. Results: We found that PHD2 accumulates in the nucleus when nuclear export is inhibited, indicating that PHD2 shuttles in and out of the nucleus. An interaction between PHD2 and the nuclear export receptor CRM1 could be confirmed, while PHD2 does not bind to any importin areceptor tested. We further showed that amino acids 191 and 192 of PHD2 play a crucial role in nuclear import and nuclear export requires amino acids 6-20. We show the effects of the localization of PHD2 on HIF-1a transcriptional activity. Conclusion: Intracellular trafficking of the HIF-1a hydroxylase PHD2 is a complex process depending on untypical structural motifs. Our data provide new insights into the regulatory mechanisms underlying the oxygen sensor cascade.