The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element (HRE). The alpha subunit of the HIF transcription factors is degraded by proteasome pathways during normoxia, but stabilized under hypoxic conditions. It has previously been established that cobalt causes accumulation of HIF-2α and HIF-1α. However, little is known about the mechanism by which cobalt mimics hypoxia and stabilizes these transcription factors. We show here that cobalt binds directly to HIF-2α in vitro with a high affinity and in an oxygen-dependent manner. We found that HIF-2α, which had been stabilized with a proteasome inhibitor, could bind to cobalt, whereas hypoxia-stabilized HIF-2α could not. Mutations within the oxygen-dependent degradation domain of HIF-2α prevented cobalt binding and led to accumulation of HIF-2α during normoxia. This suggests that transition metal such as iron may play a role in regulation of HIF-2α in vivo.