Hypoxia-inducible factor (HIF)-1α is a transcription factor that plays a critical role in regulating genes involved in erythropoiesis and angiogenesis. Recent evidence indicates that the von Hippel-Lindau tumor suppressor protein (VHL) is part of a ubiquitin ligase complex that promotes the degradation of HIF-1α under normoxic conditions. Under hypoxic conditions, HIF-1α is markedly stabilized. A critical issue in understanding the hypoxic response is the identification of hypoxia-regulated steps. We show here that hypoxia and cobalt treatment modulate the capacity of a HIF-1α fragment comprising residues 531–652 to coimmunoprecipitate with VHL. Hypoxia and cobalt both significantly diminish the interaction, and furthermore, normoxia treatment after hypoxia rapidly normalizes it. This HIF-1α fragment confers hypoxia and cobalt inducibility on a heterologous protein. Significantly, contained within this fragment is a short 27-residue sequence that behaves identically in all respects noted above. Finally, evidence is provided to show that cobalt and hypoxia both induce a posttranslational modification (or loss of one) in HIF-1α that affects its binding to VHL. We propose that dynamic, site-specific interaction of HIF-1α with VHL provides one mechanism by which HIF-1α can be regulated.