Affinity-purified rabbit polyclonal (PAB96-1) and mouse monoclonal (1G9-1C2) antibodies to synthetic H-DDDDDDD-OH, an antimicrobial anionic peptide (AP) originally isolated from ovine pulmonary surfactant, were prepared and used to assess the concentrations of AP-like molecules in human respiratory tract samples. In bronchoalveolar lavage fluids, concentrations of AP-like molecules measured by enzyme-linked immunosorbent assay were significantly lower in 13 patients with cystic fibrosis (CF) (mean ± standard deviation [SD], 0.78 ± 0.46 mM) than in 34 patients without CF (1.30 ± 0.66 mM) (P = 0.01). In pulmonary tissues of three patients without CF, very little antigen was stained in the apical cytoplasm of the bronchial and bronchiolar epithelium yet robust staining was seen in the alveolar epithelium. In pulmonary tissues of three patients with CF, robust staining of antigen was seen in the apical cytoplasm of the bronchial and bronchiolar epithelium yet no staining was seen in the alveolar epithelium. These results show that AP-like molecules are present in healthy human respiratory tract samples and differ in concentration and location of expression in patients with and without CF.