Neonates with congenital diaphragmatic hernia (CDH) continue to have unacceptably high mortality rates. To better understand the associated pulmonary pathology we measured biochemical parameters of lung maturity in neonatal rats with or without congenital diaphragmatic hernia created by maternal feeding of a single dose of nitrofen on day 9.5 or day 11.5 of gestation. Lungs from neonatal rats with large CDH (n = 9, 5 right-sided, 4 left-sided) had a significantly lower lung weight (P = .0001), lung weight/body weight ratio (P = .0001), disaturated phosphatidylcholine (DSPC) per microgram DNA (P < .005), total DSPC (P = .0001), total DNA (P < .05), protein per microgram DNA (P < .05), and total protein content (P < .005) when compared with lungs from the litter mates without congenital diaphragmatic hernia (n = 10). The lungs of rats with hernia also had significantly higher DNA concentrations (P < .05) and glycogen concentrations (P < .05). These data demonstrate that lungs in neonatal rats with large CDH are biochemically immature. Treatment directed toward correcting the pulmonary biochemical immaturity of affected fetuses before birth may improve the prognosis for these babies.