The authors describe a one-dimensional thin-layer chromatographic method for separating lecithin, sphingomyelin, phosphatidylglycerol, and other phospholipids. The occurrence of phosphatidylglycerol in relation to the lecithin/sphingomyelin ratio is reported for 261 amniotic fluid samples. This compound does not consistently appear until the ratio exceeds about 3.1, and occurs less often in samples from diabetic patients than in those from normal patients. The respiratory distress syndrome did not occur when phosphatidylglycerol was present in amniotic fluid although the reverse was not necessarily true. Thus the presence of phosphatidylglycerol offers additional assurance of pulmonary maturity.