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Objective: Treatment of preterm infants with respiratory distress syndrome (RDS) with exogenous surfactant has greatly improved clinical outcome. Some infants require multiple doses, and it has not been studied whether these large amounts of exogenous surfactant disturb endogenous surfactant metabolism in humans. We studied endogenous surfactant metabolism in relation to different amounts of exogenous surfactant, administered as rescue therapy for RDS. Design: Prospective clinical study. Setting: Neonatal intensive care unit in a university hospital. Patients: A total of 27 preterm infants intubated and mechanically ventilated for respiratory insufficiency, Interventions: Infants received a 24-hr infusion with the stable isotope [U-C-13]glucose starting 5.3 +/- 0.5 hrs after birth. The C-13-incorporation into palmitic acid in surfactant phosphatidylcholine (PG) isolated from serial tracheal aspirates was measured. Infants received either zero (n = 5), one (n = 4), two (n = 15), or three (n = 3) doses of Survanta (100 mg/kg) when clinically indicated. Measurements and Main Results: Using multiple regression analysis, the absolute synthesis rate (ASR) of surfactant PC from plasma glucose increased with 1.3 +/- 0.4 mg/kg/day per dose of Survanta (p = .007) (mean a SEM). The ASR of surfactant PC from glucose was increased by prenatal corticosteroid treatment with 1.3 +/- 0.4 mg/kg/day per dose corticosteroid (p = .004), and by the presence of a patent ductus arteriosus with 2.1 +/- 0.7 mg/kg/day (p = .01). Conclusion: These data are reassuring and show for the first time in preterm infants that multiple doses of exogenous surfactant for RDS are tolerated well by the developing lung and stimulate endogenous surfactant synthesis.