Respiratory mitochondria spontaneously undergo quantal, brief bursts of superoxide production, named “superoxide flashes”. A property common to all species and cell types examined, generation of superoxide flashes is coupled to transient opening of mitochondrial membrane permeability transition pore (mPTP) and depends on the functional integrity of electron transfer chain (ETC). Unitary properties of superoxide flashes (amplitude, duration) appear to be stereotypical, at levels from isolated mitochondria to whole organs (e.g. beating heart) and even to live animals. Functionally, superoxide flashes act as elemental reactive oxygen species (ROS) signaling events (“signaling ROS”) that participate in diverse cellular processes, whereas constitutive electron leakage from the ETC to molecular oxygen produces the bulk of ROS for the regulation of redox homeostasis (“homeostatic ROS”). In particular, superoxide flash incidence provides a digital readout to gauge the glucose- and insulin-stimulated mitochondrial metabolism in live animals, and a novel biomarker of the oxidative stress in hyperosmotic stress and ischemia-reperfusion injury. These findings support the notion that ROS act as second messengers in physiological and pathophysiological contexts, and demonstrated the paramount importance of spatiotemporal organization of ROS signals in determining their signaling efficiency and modality. Keywords: Reactive oxygen species (ROS); Superoxide flash; Local ROS signaling; Mitochondrial permeability transition pore; Mitochondria