Oxidative stresses from irritants such as hydrogen peroxide and ozone (O-3) can cause dysfunction of the pulmonary surfactant (PS) layer in the human lung, resulting in chronic diseases of the respiratory tract For identification of structural changes of pulmonary surfactant protein B (SP-B) due to the heterogeneous reaction with 03, field-induced droplet ionization (FIDI) mass spectrometry has been utilized FIDI is a soft ionization method in which ions are extracted from the surface of microliter-volume droplets We report structurally specific oxidative changes of SP-B,25 (a shortened version of human SP-B) at the air-liquid interface. We also present studies of the interfacial oxidation of SP-B1-25 in a nonionizable 1-palmitoyl-2-oleoyl-sn-glycerol (POG) surfactant layer as a model PS system, where competitive oxidation of the two components is observed. Our results indicate that the heterogeneous reaction of SP-B1-25 at the interface is quite different from that in the solution phase. In comparison with the nearly complete homogeneous oxidation of SP-B1-25, only a subset of the amino acids known to react with ozone are oxidized by direct ozonolysis in the hydrophobic interfacial environment, both with and without the lipid surfactant layer. Combining these experimental observations with the results of molecular dynamics simulations provides an improved understanding of the interfacial structure and chemistry of a model lung surfactant system subjected to oxidative stress