Multicellular organisms initiate adaptive responses when oxygen (O₂) availability decreases, but the underlying mechanism of O₂ sensing remains elusive. We find that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1α and HIF-2α and that an increase in reactive oxygen species (ROS) links this complex to HIF-α stabilization. Using RNAi to suppress expression of the Rieske iron-sulfur protein of complex III, hypoxia-induced HIF-1α stabilization is attenuated, and ROS production, measured using a novel ROS-sensitive FRET probe, is decreased. These results demonstrate that mitochondria function as O₂ sensors and signal hypoxic HIF-1α and HIF-2α stabilization by releasing ROS to the cytosol.