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- 13C 4] palmitate and [1- 13C 1] 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.