A technique using solubilization with sodium cholate to resolve 31P NMR resonances of phospholipid molecular species was applied to amniotic fluid samples from 16 subjects. Gestational ages from 25 to 40 weeks were represented, and two subjects were sampled sequentially. Fitting of the partially resolved 31P NMR signal of phosphatidylcholine (PC) generated an estimate of percent disaturated acyl PC (%dsPC) which correlated more highly with gestational age than did several other potential indices of fetal lung maturation, such as the ratio of PC to inorganic phosphate from the solubilized spectra, or PC to sphingomyelin from extract spectra. In a few cases, enzyme-catalyzed PC hydrolysis limited the precision, but did not appear to affect the accuracy, of the %dsPC estimates. Resolution of palmitoyl and oleoyl lyso-PC species was observed for both the 1- and 2-acyl isomers. Upfield shifts due to the presence of cis double bonds in the lone acyl chain of the lyso-PCs were analogous to those observed for the diacyl PCs.