The concentration and fatty acid composition of acetone- and non-acetone-precipitated lecithin, the L/S ratio, and the concentration of phosphate in lipids were measured in aliquots of amniotic fluid obtained at different periods of gestation. Irrespective of the technique of precipitation of the phospholipids, no significant differences in fatty acid composition or concentration of lecithin can be detected within the same gestational age except for the presence of C12 and C20 fatty acids in the acetone-soluble phospholipids. Increase in C16:0 fatty acid concentration and composition can be demonstrated with fetal age in the acetone- and non-acetone-precipitated lecithin. The L/S ratios measured in both preparations differ numerically, but are linearly related. The L/S ratio can be evaluated visually as well as by densitometry. The concentration of phosphate in total amniotic fluid lipid increases with gestational age, though the S.D. is large. These results demonstrate that the acetone precipitation of phospholipids with densitometry measurement of the L/S ratio offers no advantages over the visual determination of the ratio on total amniotic phospholipids. It also indicates that the acetone precipitation of lecithin is not specific for lung surfactant, at least in amniotic fluid. There is no evidence that lecithin synthesized in the second trimester contains a high percentage of C14 chain length fatty acids, as would be expected if the methylation pathway was predominant at this stage of gestation.