While cellular responses to low oxygen (O₂) or hypoxia have been studied extensively, the precise identity of mammalian cellular O₂ sensors remains controversial. Using murine embryonic cells lacking cytochrome c, and therefore mitochondrial activity, we show that mitochondrial reactive oxygen species (mtROS) are essential for proper O₂ sensing and subsequent HIF-1α and HIF-2α stabilization at 1.5% O₂. In the absence of this signal, HIF-α subunits continue to be degraded. Furthermore, exogenous treatment with H₂O₂ or severe O₂ deprivation is sufficient to stabilize HIF-α even in the absence of cytochrome c and functional mitochondria. These results provide genetic evidence indicating that mtROS act upstream of prolyl hydroxylases in regulating HIF-1α and HIF-2α in this O₂-sensing pathway.