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Directional movement of fibroblasts is a critical step in tissue repair and healing. PDGF and serum-derived cytokines are the major chemoattractants that navigate fibroblasts into wounds, but little is known how the external information is transmitted into these cells and what molecules mediate the directional sensing. In acute wounds hydrogen peroxide (H2O2) forms tissue-scale gradients and recruits the early leukocytes into these site. Whether H2O2 acts as a chemoattractant or intracellular signaling molecule during fibroblast migration is not known. Here we used biosensor approach to explore microscale patterns of H2O2 in live fibroblasts and to investigate how the internal H2O2 affects fibroblast motility. We developed a method of long-time (8-24 h) intravital registration of intracellular H2O2 patterns during fibroblast migration. We observed that intracellular gradients of H2O2 always oriented in the direction of cell locomotion under a variety of conditions, including the external chemotactic gradients, uniform stimulation and random motility in the absence of serum. Using double sensor technique we find that intracellular gradients of H2O2 closely follow those of PIP3, a well established chemotactic molecule. We further demonstrate that PI3-kinase/PIP3 functions upstream of H2O2 generation, but not H2O2 gradient formation. If PIP3 regulates both speed and directionality of migration, H2O2 gradient controls only speed component by protrusion stabilizing. We conclude that H2O2 is critical activator of fibroblast motility rather than compass molecule.