The transcriptional activator complex HIF-1 plays a key role in the long term adaptation of cells and tissues to their hypoxic microenvironment by stimulating the expression of genes involved in angiogenesis and glycolysis. The expression of the HIF-1 complex is regulated by the levels of its HIF-alpha subunits that are degraded under normoxic conditions by the ubiquitin-proteasome system. Whereas this pathway of HIF-alpha protein degradation has been well characterized, little is known of their turnover during prolonged hypoxic conditions. Herein, we describe a pathway by which HIF-1alpha and HIF-2alpha proteins are constitutively degraded during hypoxia by the proteasome system, although without requirement of prior ubiquitylation. The constitutive/hypoxic degradation of HIF-alpha proteins is independent of the presence of VHL, binding to DNA, or the formation of a transcriptionally active HIF-1 complex. These results are further strengthened by the demonstration that HIF-alpha proteins are directly degraded in a reconstituted in vitro assay by the proteasome. Finally, we demonstrate that the persistent down-regulation of HIF-1alpha during prolonged hypoxia is mainly caused by a decreased production of the protein without change in its degradation rate. This constitutive, ubiquitin-independent proteasomal degradation pathway of HIF-alpha proteins has to be taken into account in understanding the biology as well as in the development of therapeutic interventions of highly hypoxic tumors.