We describe an indirect test of fetal lung maturity: the quantitation of disaturated phosphatidylcholine in amniotic fluid. The lipids in samples of amniotic fluid from 172 patients were reacted with osmium tetroxide, and disaturated phosphatidylcholine was then isolated by thin-layer chromatography. Interfering substances were retained by a pre-adsorbent layer. The charred disaturated phosphatidylcholine, quantitated by densitometry, was compared to standard dipalmitoyl phosphatidylcholine. Both within-run and between-run coefficients of variation were about 10%. Blood and meconium do not interfere. Six infants developed respiratory distress when disaturated phosphatidylcholine concentrations of amniotic fluid drawn within 72 h of delivery were <5.5 mg/L. A concurrently determined lecithin/sphingomyelin ratio falsely predicted lung maturity in one of these. In seven other samples for which lecithin/sphingomyelin ratios suggested lung immaturity but disaturated phosphatidyl-choline predicted maturity, none of the infants developed respiratory distress. In normal pregnancies, measurement of disaturated phosphatidylcholine in amniotic fluid appears to be a better predictor of fetal lung maturity than is measurement of the lecithin/sphingomyelin ratio. Further studies are needed to determine if this analysis is a better predictor in diabetic pregnancies.