We have developed a high-performance liquid chromatographic (HPLC) method for the analyses of surface-active amniotic fluid phospholipids, lecithin (L), sphingomyelin (S), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS), which are important in the prediction of fetal lung maturity. The method incorporates an internal standard in the amniotic fluid extract, and utilizes a 10-microliter aliquot of a 2:1 chloroform-methanol extract of amniotic fluid injected onto a 5-micron DIOL or CN HPLC column, and a variable-wavelength detector set at 203 nm. Amniotic fluid phospholipid estimations were determined on 40 amniotic fluid samples by the HPLC method and by the routine thin-layer chromatographic (TLC) method. Good agreement was observed between the two methods for the L/S ratio, PG, and PI (rPG 0.94, rPI 0.95, rL/S 0.97). The advantages of the HPLC procedure include: Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. The internal standard allows individual concentration of phospholipids to be estimated. The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.