Emerging evidence suggests that reactive oxygen species (ROS) constitute a class of signaling molecules that regulate diverse cell functions including metabolism, muscle contractility and apoptosis. Recently we have developed and characterized a highly sensitive and reversible superoxide-selective probe, a circularly permuted yellow fluorescent protein (cpYFP), and demonstrated quantal and transient superoxide-producing events (superoxide flashes) within single mitochondria across multiple cell types (Wang et al, Cell, 132, 279). To further understand the physiological significance of flash events, we generated the pan-tissue mt-cpYFP transgenic mice expressing cpYFP in the mitochondria of multiple tissues. In vivo imaging of superoxide signals in gastrocnemius of transgenic mouse under anesthesia revealed mitochondrial superoxide flashes with similar properties (Fig). Further, superoxide flashes were also visualized in isolated skeletal muscle fibers transfected in vivo by electroporation with mt-cpYFP. Our findings support that mitochondrial superoxide flash activity is a physiologically relevant phenomenon that may participate in diverse aspects of cell function and signaling.