Hydrogen peroxide (H2O2) is known to play an important role in airway homeostasis. For this reason its levels and thus its synthesis and consumption are important mechanisms for controlling airway functions. We have identified the major macromolecular consumer of H2O2 in sheep airway secretions to be lactoperoxidase (LPO), a heme peroxidase previously studied in milk and saliva. This enzyme uses H2O2 to oxidize the anion thiocyanate to an antibiotic compound that prevents growth of bacteria, fungi, and viruses. LPO was isolated from sheep airways and proved to be a major constituent comprising about 1% of the soluble protein in airway secretions. The isolated airway LPO was catalytically active and displayed the enzymatic characteristics previously described for the enzyme isolated from bovine milk. Airway LPO activity was shown to increase the rate of bacterial clearance from sheep airways. The role of this enzyme in the airway host defense strongly suggests that an active H2O2 production system exists to supply appropriate substrate for the enzyme. The identity of this H2O2 synthesis system is an important, yet unknown feature of airway oxygen radical metabolism.