The purposes of this study were to adapt and evaluate further a pulmonary surfactant isolation method applicable to unperfused fetal rat lung, to quantitate key phospholipids phosphatidylcholine (disaturated phosphatidylcholine, and phosphatidylglycerol) of the isolated material during the last 3 days of gestation, and to determine if abnormalities in surfactant phospholipids were present in fetuses of diabetic pregnancies. A simplified scheme of sucrose gradient centrifugation proved useful for small scale preparations of material enriched in the phospholipids most characteristic of pulmonary surfactant. It was shown that fetal blood phospholipids did not contaminate the surfactant fraction and therefore would not produce artifacts in assessment of lung maturational changes. Analyses of subcellular fractions isolated at 19.5, 20.5, and 21.5 days revealed that the percentages of disaturated phosphatidylcholine relative to total phospholipids were 23-44% in the surfactant preparations and 14-21% in the residual (nonsurfactant) fractions, while the disaturated phosphatidylcholine/phosphatidylcholine ratios were 0.62 +/- 0.06 and 0.41 +/- 0.03, respectively. Summation of the amounts of individual phospholipids in the two fractions yielded data that were nearly identical to the concentrations of these compounds in whole fetal lung samples analyzed independently, implying that losses during the surfactant isolation technique were negligible. The concentrations of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, and total phospholipids increase markedly (more than 10-fold) and progressively in surfactant fractions prepared from normal fetal rat lung at 19.5, 20.5, and 21.5 days of gestation. In contrast, the residual fractions showed no changes from 19.5 to 20.5 days and then relatively modest increases from 20.5 to 21.5 days, except for phosphatidylglycerol, which increased markedly.(ABSTRACT TRUNCATED AT 250 WORDS)