Oxygen‐dependent proteolytic destruction of hypoxia‐inducible factor‐α (HIF‐α) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel–Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF‐1α that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF‐α oxygen‐dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non‐redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL–HIF‐α interactions, but reinforces the role of prolyl hydroxylation as an oxygen‐dependent destruction signal.