In animal CDH models, surfactant deficiency contributes to the pathophysiology of the condition but information on human disease is very limited. The aim of our study was to investigate surfactant kinetics in CDH newborns. We studied surfactant disaturated-phosphatidylcholine (DSPC) half-life, turnover and apparent pool size by stable isotope methodology in CDH newborns with no ExtraCorporeal Membrane Oxygenation (ECMO) support (n= 13, birth weight (BW) 3.2 ± 2.2 kg, gestational age (GA) 39 ± 0.4 wks, postnatal age 43 ± 11 h) and in 8 term infants with no lung disease (CONTROLS, BW 2.7 ± 0 kg, GA 38 ± 0.8 wks, postnatal age 96 ± 26 h). We administered a trace dose of13C-palmitic acid dipalmitoyl-phosphatidylcholine (DPPC) through the endotracheal (ET) tube and we measured DSPC kinetics by gas chromatography-mass spectrometry from DSPC13C-enrichment decay curves obtained from sequential tracheal aspirates. DSPC amount from tracheal aspirates (TA-DSPC) was measured by gas chromatography. In CDH infants DSPC half-life was shorter (24 ± 4 and 53 ± 11 h,p= 0.01), turnover faster (0.6 ± 0.1 and 1.5 ± 0.3 d−1p= 0.01), apparent pool size smaller (34 ± 6 and 57 ± 7 mg/kg body weight,p= 0.02) and tracheal aspirates DSPC amount lower (2.4 ± 0.4 and 4.6 ± 0.5 mg/mL Epithelial Lining Fluid (ELF),p= 0.007) than in CONTROLS. In conclusion surfactant kinetics is grossly abnormal in mechanically ventilated CDH. Whether alterations of DSPC kinetics in CDH infants are caused by a primary surfactant deficiency or are secondary to oxygen therapy and ventilator support has still to be determined.