Mammalian cells detect decreases in oxygen concentrations to activate a variety of responses that help cells adapt to low oxygen levels (hypoxia). One such response is stabilization of the protein HIF-1α, a component of the transcription factor HIF-1. Here we show that a small interfering RNA (siRNA) against the Rieske iron-sulfur protein of mitochondrial complex III prevents the hypoxic stabilization of HIF-1α protein. Fibroblasts from a patient with Leigh's syndrome, which display residual levels of electron transport activity and are incompetent in oxidative phosphorylation, stabilize HIF-1α during hypoxia. The expression of glutathione peroxidase or catalase, but not superoxide dismutase 1 or 2, prevents the hypoxic stabilization of HIF-1α. These findings provide genetic evidence that oxygen sensing is dependent on mitochondrial-generated reactive oxygen species (ROS) but independent of oxidative phosphorylation.