Despite their structural similarity, hypoxia-inducible factor (HIF)-1 alpha and HIF-2 alpha have distinct functional properties and exhibit distinct spatiotemporal expression patterns, suggesting that the expressions of the two proteins are regulated by different mechanisms. To clarify the HIF-2 alpha-specific regulatory mechanism, we screened HIF-2 alpha-associated proteins in a yeast two-hybrid system and identified kelch-like 20 (KLHI20). HIF-2 alpha, but not HIF-1 alpha, interacted with KLHL20. siRNA-mediated knockdown of KLHL20 decreased HIF-2 alpha protein, but not HIF-2 alpha mRNA or HIF-1 alpha protein. Depletion of KLHL20 decreased hypoxia-induced HIF activity, and consequently resulted in decreased expression levels of HIF-2 alpha-responsive genes such as VEGF and CITED2. In contrast, overexpression of KLHL20 increased the expression levels and transcriptional activities of the O(2)-sensitive wild-type and O(2)-insensitive mutant forms of HIF-2 alpha. KLHL20 siRNA also inhibited HIF-2 activity in von Hippel-Lindau tumor suppressor protein (pVHL)-deficient 786-O cells. These results indicate that KLHL20 is a novel player that regulates HIF-2 alpha protein expression through mechanisms independent of hypoxia and pVHL. (C) 2011 Elsevier Inc. All rights reserved.