Recent efforts have been aimed at targeting the hypoxia inducible factor (HIF)-mediated hypoxia-induced gene pathway for renal cell carcinomas (RCC) therapy. Among the various genes induced by HIF, vascular endothelial growth factor (VEGF) is one of the critical mediators in angiogenesis, tumor growth and metastasis. To date, however, limited information is available on the functional differences regarding VEGF transcription between the HIF subunits, namely HIF-1α and HIF-2α. To investigate the HIF-1α and HIF-2α-dependent effect on VEGF gene induction in RCC, a panel of human RCC cell lines was analyzed. We found that a loss of HIF-1α protein expression was a common event in RCC cell lines, which was associated not only with truncated HIF-1α mRNA transcripts but also with transcriptional silencing. Since the CpG rich promoter region of the HIF-1α gene contained a similar frequency of methylated CpG dinucleotides in RCC cell lines, a complex and non-uniform mechanism may be involved in this phenomenon. In these HIF-1α defective cell lines, the knockdown of the HIF-2α gene demonstrated that HIF-2α regulated the VEGF production, irrespective of the VHL gene mutation status. In contrast, HIF-1α played a predominant role in VEGF secretion in the cells expressing both wild-type HIF-1α and HIF-2α proteins. HIF-1α may therefore represent an important target molecule for RCC therapy; however, HIF-2α should be targeted in HIF-1α defective renal cancer cells.