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-2 alpha and HIF-1 alpha. 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-2 alpha in vitro with a high affinity and in an oxygen-dependent manner. We found that HIF-2 alpha, which had been stabilized with a proteasome inhibitor, could bind to cobalt, whereas hypoxia-stabilized HIF-2 alpha could not. Mutations within the oxygen-dependent degradation domain of HIF-2 alpha prevented cobalt binding and led to accumulation of HIF-2 alpha during normoxia. This suggests that transition metal such as iron may play a role in regulation of HIP-2 alpha in vivo.