The hypoxia-inducible factor-1alpha (HIF-1alpha) is a master regulator of the cellular response to decreased oxygen levels. This transcription factor is highly unstable at normal oxygen concentrations and is rapidly stabilized by hypoxia. At normoxia two specific proline residues (Pro(402) and Pro(563)) of mHIF-1alpha are hydroxylated and recognized by the von Hippel-Lindau E3 ubiquitin ligase (pVHL) complex, which upon binding mediates degradation of the protein. Previous studies have demonstrated that these two proline residues are critical for high affinity binding to pVHL. We have performed a detailed analysis of a mutant form of HIF-1alpha, where both these proline residues have been mutated, and we have uncovered a novel degradation pathway, to which the HIF-1alpha mutant protein is not resistant. Our results show that the HIF-1alpha double proline mutant undergoes ubiquitination and proteasome-dependent degradation, and retains the ability to be stabilized in response to hypoxia and CoCl(2) treatment. However in contrast to the wild-type protein, stabilization of the mutant was only observed within short periods of hypoxia exposure (1-2 h). Degradation assays in the presence of the expressed prolyl hydroxylases (PHDs) 1-3 showed that, unlike the wild-type protein, the HIF-1alpha mutant was resistant to these hydroxylases. However, experiments knocking-down expression of pVHL by RNA interference showed that the HIF-1alpha mutant is degraded and ubiquitinated by a pVHL-mediated mechanism. In conclusion, we show the first evidence of a novel mechanism of degradation of HIF-1alpha at normoxia that involves pVHL but is not mediated by PHDs 1-3 or by degradation boxes surrounding Pro(402) and Pro(563).