OBJECTIVE The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect. METHODS HIF-1alpha protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O(2), 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1alpha mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR). RESULTS Both inhibitors drastically reduced hypoxic HIF-1alpha accumulation (2-DG + hypoxia 2% mean HIF-1alpha protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1alpha protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1alpha mRNA levels, suggesting a translational or posttranslational mechanism. CONCLUSIONS The effect of glycolysis modulation on the HIF-1alpha levels in tumor cells may provide a novel approach to therapeutically target HIF-1alpha.