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Hydrogen peroxide (H2O2) is found in exhaled breath and is produced by airway epithelia. In addition, H2O2 is a necessary substrate for the airway lactoperoxidase (LPO) anti-infection system. To investigate the source of H2O2 produced by airway epithelia, PCR was used to screen nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in human airway epithelia redifferentiated at the air-liquid interface (ALI) and demonstrated the presence of Duox1 and 2. Western blots of culture extracts indicated strong expression of Duox, and immunohistochemistry of human tracheal sections localized the protein to the apical portion of epithelial cells. Apical H2O2 production was stimulated by 100 μM ATP or 1 μM thapsigargin, but not 100 μM ADP. Diphenyleneiodonium, an NADPH oxidase inhibitor, and dimethylthiourea, a reactive oxygen species scavenger, both inhibited this stimulation. ATP did not stimulate the basolateral H2O2 production by ALI cultures. ATP and thapsigargin increased intracellular Ca2+ with kinetics similar to increasing H2O2 production, and thus consistent with the expected Ca2+ sensitivity of Duox. These data suggest that Duox is the major NADPH oxidase expressed in airway epithelia and therefore a contributor of H2O2 production in the airway lumen. In addition, the data suggest that extracellular H2O2 production may be regulated by stimuli that raise intracellular Ca2+.