Objectives: To use stable isotopically labeled precursors of pulmonary surfactant phospholipids to measure precursor utilization and surfactant turnover in premature infants who required mechanical ventilation at birth, 2 weeks, and >4 weeks of age. Study design: Infants of ≤28 weeks' gestation received simultaneous 24-hour intravenous infusions of [1,2,3,4- 13C4] palmitate and [1-13C1] acetate at birth, 2 weeks, and ≥4 weeks of life. Disaturated phospholipids were extracted from sequential tracheal aspirate samples obtained over a period of 2 weeks. Fractional catabolic rate (a measure of total turnover) and the fractional synthetic rates from plasma palmitate and de novo synthesis (acetate) were measured. Results: The fractional catabolic rate increased from 25.3% ± 7.0% per day at birth to 53.8% ± 14.4% per day at 4 weeks (P = .001). The combined contribution from plasma palmitate and de novo synthesis to total synthesis increased from 44.2% ± 19.8% at birth to 85.2% ± 32.8% at 4 weeks (P = .03). Conclusions: Total surfactant turnover increased in premature infants with evolving bronchopulmonary dysplasia. The increasing contributions from acetate and plasma palmitate suggest a decrease in surfactant phospholipid recycling.