In normoxic cells the hypoxia‐inducible factor‐1α (HIF‐1α) is rapidly degraded by the ubiquitin‐proteasome pathway, and activation of HIF‐1α to a functional form requires protein stabilization. Here we show that the product of the von Hippel‐Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF‐1α under normoxic conditions via interaction with the core of the oxygen‐dependent degradation domain of HIF‐1α. The region of VHL mediating interaction with HIF‐1α overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF‐1α and subsequent degradation. Interestingly, the VHL binding site within HIF‐1α overlapped with one of the minimal transactivation domains. Protection of HIF‐1α against degradation by VHL was a multistep mechanism, including hypoxia‐induced nuclear translocation of HIF‐1α and an intranuclear hypoxia‐dependent signal. VHL was not released from HIF‐1α during this process. Finally, stabilization of HIF‐1α protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.