We have developed enzyme-linked immunoassays (ELISAs) that measure major proteins that are associated with pulmonary surfactant. Using these ELISAs, we tested sequential tracheal aspirates from infants severely ill with respiratory distress syndrome (RDS) who had been treated either with exogenous surfactant or with placebo within 8 h of birth. On average, we found low concentrations of surfactant proteins in tracheal aspirates on Day 1 of life, with increases evident by Day 3. The surfactant used in this study (TA surfactant) contains only the low molecular weight (6 kDa) surfactant proteins and not the 35 kDa surfactant protein. As we expected, those who were treated with TA surfactant more frequently had detectable concentrations of low molecular weight surfactant protein on the second day of life when compared with control infants. No differences were evident in the concentrations of surfactant proteins between the 2 groups by Day 3, nor were differences evident between the 2 groups evident for 35 kDa surfactant protein during the first 3 days of life. Increased low molecular weight surfactant proteins in tracheal aspirates 1 to 2 days after surfactant therapy may occur either because of persistence of exogenous surfactant proteins and/or enhanced surfactant protein production. Comparisons with measurements from other groups of patients with RDS confirm that absence of both surfactant proteins reflects alveolar surfactant deficiency.