The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1-3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography-tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.