The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity usingN-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H₂O₂. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. The reaction stoichiometry of Amplex Red and H₂O₂was determined to be 1:1. This probe allows detection of 5 pmol H₂O₂in a 96-well fluorescence microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H₂O₂release from as few as 2000 phorbol myristate acetate-stimulated neutrophils with a sensitivity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catalyzed assay using Amplex Red results in anincreasein fluorescence on oxidation rather than adecreasein fluorescence as in the scopoletin assay. In comparison with other fluorometric and spectrophotometric assays for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity and specificity, Amplex Red should have a broad application for the measurement of H₂O₂in a variety of oxidase-mediated reactions and very low levels of H₂O₂in food, environmental waters, and consumer products.