In the present study, the role of the C-terminal alpha-helical domain (amino acid (aa) 195-208) of the von Hippel-Lindau (VHL) tumour suppressor was investigated. Deletions of the VHL C-terminus up to the naturally occurring 195-Gln-Term resulted in hypoxia-inducible factor (HIF)-1alpha downregulation in renal cell carcinoma (RCC)4 cells during normoxia, suggesting that this domain is not an absolute requirement for the ubiquitination of HIF-1alpha. However, detailed investigation of the ubiquitin protein isopeptide ligase ubiquitin ligase properties of VHL revealed C-terminal deletions to cause a significant impairment of HIF-1alpha ubiquitination, which is shown to be due to a loss in high-affinity binding to the target substrate. When VHL regulation of both HIF-1alpha N- and C-terminal oxygen-dependent degradation domains (HIF-ODDD) was investigated, it was found that only ubiquitination of the C-terminal HIF-ODDD was affected by the deletion of the VHL C-terminus. When RCC4 cells expressing C-terminal truncations of VHL were exposed to graded hypoxia, differences in the induction of HIF-1alpha were observed in comparison with full-length VHL, with a shift in the maximal induction of HIF-1alpha to a higher oxygen tension. These changes were accompanied by increased glucose transporter 1 expression, p300 CH1 domain binding and HIF-mediated reporter activity. We have thus defined a role for the C-terminal alpha-helical domain of VHL in the regulation of HIF-1alpha.