Abstract Background Decreased expired nitric oxide (eNO) is commonly observed in cystic fibrosis (CF) patients and is usually explained by dysregulation of NO synthase (NOS) isoforms in respiratory tract epithelium. Later stages of this disease are accompanied by intense airway infiltration of phagocytes with high NOS activity, abundant levels of the hemoprotein myeloperoxidase (MPO) and significant production of significant reactive oxygen species. Methods This study characterizes the contribution of the high airway levels of MPO to decreased eNO levels in adult CF patients. NO metabolites (NO x ) and MPO levels in fresh sputum of control and adult CF patients were determined and related to measurements of eNO and to in vitro consumption of NO in CF sputum. Results Despite essentially equal levels of NO x in sputum, eNO was 2- to 3-fold lower in CF patients compared to healthy controls. In CF patients, eNO levels were negatively associated with sputum peroxidase activity. In vivo correlations were confirmed by ex vivo studies of NO consumption by MPO in CF sputum. Immunodepletion studies confirmed MPO as the major heme peroxidase in CF sputum contributing to the hydrogen peroxide (H 2O 2)-dependent consumption of NO. Conclusions In CF airways MPO acts as a phagocyte-derived NO oxidase that diminishes NO bioavailability at airway surfaces, possibly identifying this peroxidase as a potential target for therapeutic intervention.