SummaryMale infants have a higher risk of respiratory distress syndrome than females at concurrent gestations. Recent evidence in humans has linked fetal sex with differences in amniotic fluid indices of lung maturation. We tested the hypothesis that the late gestation surge in pulmonary surfactant production occurs later in the male fetus than in the female fetus in the rabbit model. We measured saturated phosphatidylcholine and total phosphatidylcholine in lung lavage at 26, 28, and 30 days gestation and in amniotic fluid at 24, 26, 28, and 30 days gestation (term = 31 days). The saturated phosphatidylcholine/sphingomyelin ratios were 158 and 55% higher in female fetal lung lavage at 26 and 28 days, respectively, and 75% higher in amniotic fluid at 28 days (P< 0.05). The total phosphatidylcholine/sphingomyelin ratios were 39% higher in female fetal lung lavage and 35% higher in female amniotic fluid at 28 days (P< 0.05). Significant differences were not detected in the very immature (24 day) or the mature (30 day) fetuses. This provides clear evidence of a biochemical difference according to fetal sex in the maturation of pulmonary surfactant production.