The glutathione redox couple (GSH/GSSG) and hydrogen peroxide (H(2)O(2)) are central to redox homeostasis and redox signaling, yet their distribution within an organism is difficult to measure. Using genetically encoded redox probes in Drosophila, we establish quantitative in vivo mapping of the glutathione redox potential (E(GSH)) and H(2)O(2) in defined subcellular compartments (cytosol and mitochondria) across the whole animal during development and aging. A chemical strategy to trap the in vivo redox state of the transgenic biosensor during specimen dissection and fixation expands the scope of fluorescence redox imaging to include the deep tissues of the adult fly. We find that development and aging are associated with redox changes that are distinctly redox couple-, subcellular compartment-, and tissue-specific. Midgut enterocytes are identified as prominent sites of age-dependent cytosolic H(2)O(2) accumulation. A longer life span correlated with increased formation of oxidants in the gut, rather than a decrease.