By TLC-immunostaining with the monoclonal antibody VJ-41, which preferentially reacted with sphingomyelin (Sm) and disaturated fatty acid-containing phosphatidyl choline (DSPC), Sm and surfactant phospholipid dipalmitoyl PC were only detected in the lipid extracts from human amniotic fluid. The method was useful in the selective and simultaneous determination of surfactant phospholipid and Sm concentrations in the amniotic fluids to determine the level of maturity of the lungs of the fetus. By measuring the density of spots visualized by TLC-immunostaining, we detected Sm at a sensitivity two times higher than that for dipalmitoyl PC using the antibody. More than 50 ng of dipalmitoyl PC and Sm was detected on the same TLC plate and the standard curves were linear up to 1 microgram of phospholipids. The method was applied to determine the surfactant phospholipid/Sm ratio in 20 microliter of the amniotic fluids obtained at delivery, and the amniotic fluids from the women who delivered a baby suffering from respiratory distress syndrome (RDS) were easily discriminated from the normal amniotic fluids. In an analysis of 200 microliter of amniotic fluids from 4 RDS cases and 16 normal baby cases, the mean DSPC/Sm ratios were 0.97 +/- 0.53 and 5.75 +/- 1.29, respectively.