Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO-catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant-generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1-dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO-mediated antimicrobial defense mechanisms on mucosal surfaces.