The hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1 alpha(-/-) and hif-2 alpha(-/-) mice, in their functions. This implies that HIF-1 alpha and HIF-2 alpha may have unique target genes. To address this discrepancy and identify HIF-2 alpha-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2 alpha regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2 alpha (but not HIF-1 alpha or HIF-3 alpha) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2 alpha degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia- and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-Delta C increased endogenous HIF-2 alpha expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2 alpha-dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.