Chronic bacterial colonization of the lungs, with an excessive inflammatory response, is the major cause of morbidity and mortality in cystic fibrosis. Lung surfactant exhibits a spectrum of potential immunomodulatory properties: phospholipid components inhibit cellular inflammatory responses, whereas the hydrophilic surfactant proteins A (SP-A) and D (SP-D) are integral components of the innate host defense response of the lungs against bacterial infection. Consequently, alteration to the relative proportions of lung surfactant components may alter the susceptibility of the lungs to bacterial colonization. In this study, bronchoalveolar lavage (BAL) samples were collected at diagnostic fiberoptic bronchoscopy from 11 control children, 13 children with cystic fibrosis, and 11 children with acute lung infection. Electrospray ionization mass spectrometry analysis demonstrated negligible changes to the molecular species or total BAL concentrations of phosphatidylcholine, phosphatidylglycerol, or phosphatidylinositol among the three subject groups. In contrast, median SP-A concentration was decreased (P < 0.001) in the cystic fibrosis group (2.65 μg/ml) compared with control (12.35 μg/ml) and infection (9.76 μg/ml) groups. Median SP-D was also decreased (P < 0.05) in the infection (12.17 ng/ml) compared with the control group (641 ng/ml), and was below assay limits for the majority of cystic fibrosis children (P < 0.001). This dramatic decrease of hydrophilic surfactant proteins in the presence of normal surfactant phospholipid may be one mechanism underlying the relative ineffectiveness of the cellular inflammatory response in killing invading bacteria in the lungs of patients with cystic fibrosis.