To understand the role of hypoxia-inducible factor (HIF)-2alpha in regulating sensitivity of renal cancer cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, we transfected wild-type and mutant von Hippel Lindau (VHL) proteins into TRAIL-sensitive, VHL-negative A498 cells. We find that wild-type VHL, but not the VHL mutants S65W and C162F that do not degrade HIF proteins, cause TRAIL resistance. Knock down of the HIF-2alpha protein by RNA interference (short hairpin RNA) blocked TRAIL-induced apoptosis, decreased the level of TRAIL receptor (DR5) protein and inhibited the transcription of DR5 messenger RNA. By using luciferase constructs containing the upstream region of the DR5 promoter, we demonstrate that HIF-2alpha stimulates the transcription of the DR5 gene by activating the upstream region between -448 and -1188. Because HIF-2alpha is thought to exert its effect on gene transcription by interacting with the Max protein partner of Myc in the Myc/Max dimer, small interfering RNAs to Myc were used to lower the levels of this protein. In multiple renal cancer cell lines decreasing the levels of Myc blocked the ability of HIF-2alpha to stimulate DR5 transcription. PS-341 (VELCADE, bortezomib), a proteasome inhibitor used to treat human cancer, increases the levels of both HIF-2alpha and c-Myc and elevates the level of DR5 in renal cancer, sensitizing renal cancer cells to TRAIL therapy. Similarly, increasing HIF-2alpha in prostate and lung cancer cell lines increased the levels of DR5. Thus, in renal cancer cell lines expressing HIF-2alpha, this protein plays a role in regulating the levels of the TRAIL receptor DR5.