The influence of hormones on the synthesis of phospholipids of lung lamellar bodies was studied using lung tissue explants from 16- to 22-week gestational age human abortuses. Lung explants were incubated in a defined medium containing either no hormonal additions or insulin (2.5 micrograms/ml), cortisol (0.2 microgram/ml), or PRL (2.5 micrograms/ml), added alone or in various combinations. After 7 days of incubation, a purified lamellar body fraction was isolated from the explants. The effects of various hormone combinations on the relative rates of synthesis of lamellar body anionic glycerophospholipids during the last 24 h of culture were analyzed and compared to that of control explants. In control explants, 7.1% of the glycerophospholipid was synthesized as phosphatidylinositol, and 2.2% was synthesized as phosphatidylglycerol. Phosphatidylinositol synthesis was decreased significantly in explants incubated with cortisol alone, insulin plus cortisol, or insulin plus cortisol plus PRL (to 4.4%, 4.3%, and 3.7%, respectively), while phosphatidylglycerol synthesis was increased significantly in lamellar bodies from explants incubated with insulin plus cortisol or insulin plus cortisol plus PRL (to 5.5% and 5.7%, respectively). Although the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were significantly altered by hormones, the percentages of the total anionic glycerophospholipid were similar in all treatment groups (9.5%). The results of this study, therefore, are suggestive that lamellar body formation and glycerophospholipid composition are under hormonal control.