Summary: Several properties of a pellet fraction obtained on centrifuging amniotic fluid at 10,000 x g for 20 min were investigated. From these analyses, we defined a developmental profile which appears to describe the maturational process of the fetal lung surfactant system. At 14 to 18 wk gestation, the pellet fraction consisted of membrane-bound vesicles without internal lamellae. The phospholipid composition did not resemble that of surfactant, the major phospholipid being sphingomyelin. This stage, designated as presurfactant, persisted until 30 to 32 wk gestation. After this time, the phospholipid concentration of the pellet fraction increased continuously throughout development, and gradual but continuous changes in phospholipid composition were observed. Lecithin and phosphatidylinositol increased between 39 and 35 wk gestation. Interruption of pregnancies at this stage, termed onset of surfactant synthesis, resulted in 100% incidence of respiratory distress syndrome. From 36 wk gestation to postterm, the pellet fraction contained structures with the characteristic morphology of the lamellar inclusion bodies. The presence of single membrane components in these preparations did not contribute to the phospholipid composition. Early formed lamellar bodies lacked phosphatidylglycerol and had a high content of phosphatidylinositol. Once phosphatidylglycerol appeared in the lamellar body fraction, it continued to increase, accompanied by a decrease in phosphatidylinositol with little change in lecithin. A phosphatidylglycerol value of greater than 1% of the total phospholipids appeared to represent the stage of maturity at which there was no risk of respiratory distress syndrome. Speculation: The characterization of a pellet fraction obtained from human amniotic fluid led to the description of a developmental profile which appears to define the individual stages involved in the biochemical maturation of the fetal lung surfactant system. The development of respiratory distress syndrome appears to be very much dependent on the stage at which this process is interrupted. It is expected that such analyses can be adapted to provide a simple, yet specific index of fetal pulmonary maturity.