Activation of the hypoxia‐inducible factor α‐subunits, HIF‐1α and HIF‐2α, seems to be subject to similar regulatory mechanisms, and transgene approaches suggested partial functional redundancy. Here, we used RNA interference to determine the contribution of HIF‐1α vs. HIF‐2α to the hypoxic gene induction. Surprisingly, most genes tested were responsive only to the HIF‐1α siRNA, showing no effect by HIF‐2α knock‐down. The same was found for the activation of reporter genes driven by hypoxia‐responsive elements (HREs) from the erythropoietin (EPO), vascular endothelial growth factor, or phosphoglycerate kinase gene. Interestingly, EPO was the only gene investigated that showed responsiveness only to HIF‐2α knock‐down, as observed in Hep3B and Kelly cells. In contrast to the EPO‐HRE reporter, the complete EPO enhancer displayed dependency on HIF‐2α regulation, indicating that additional cis‐acting elements confer HIF‐2α specificity within this region. In 786‐0 cells lacking HIF‐1α protein, the identified HIF‐1α target genes were regulated by HIF‐2α. Overexpression of the HIFα subunits in different cell lines also led to a loss of target gene specificity. In conclusion, we found a remarkably restricted target gene specificity of the HIFα subunits, which can be overcome in cells with perturbations in the pVHL/HIF system and under forced expression.