A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.