Hypoxia-inducible factor-1 alpha (HIF-1alpha) constitutes a regulatory subunit of HIF-1, a major transcriptional activator of genes that coordinate physiological and pathological responses towards hypoxia. In order to identify novel interaction partners of HIF-1alpha we have applied T7 phage display system and identified a domain inherent in the retinoblastoma protein (pRB). The interaction between pRB and HIF-1alpha was confirmed by in vitro experiments and in transfected cells. Thereby, an HIF-1alpha domain spanning amino acids 530-694 was mapped to be required for pRB binding. Overexpression of pRB provoked transcriptional activation of HIF-1alpha under normoxia. Furthermore, the domain of pRB identified to bind HIF-1alpha in vitro is sufficient to cause HIF-1alpha transcriptional activation with the further notion that phosphorylation deficient pRB shows stronger HIF-1alpha transactivation. Using ChIP analysis, we show that HIF-1alpha responsive elements (HREs) are precipitated using alpha-pRB antibodies. Additionally, a functional interaction between pRB and HIF-1alpha is confirmed by showing that HIF-1alpha reverses the transcription repressor function of pRB.