Reactive oxygen species (ROS) produced by the mitochondrial electron transport chain (ETC) are widely implicated in disease etiology. However, recent evidence suggests that mitochondrial ROS are essential for normal cell function and second messengers in autophagy, apoptosis, cellular differentiation, and metabolic adaptation. Redox modification of particular cysteine sensors in proteins may mediate ROS-regulated cellular signaling in response to diverse stimuli. We hypothesize that distinct sources of mitochondrial ROS potentiate unique patterns of redox regulated cysteines which drive divergent downstream phenotypes. Specific combinations of ETC substrates and inhibitors can selectively produce ROS from the redox centers of ETC complexes I, II, and III in isolated rat skeletal muscle mitochondria and we have uncovered novel cysteines that are redox regulated by distinct sites of mitochondrial ROS production.