Phosphatidylcholine (PC) synthesis in cultured fetal rabbit lung cells was studied after exposure to various pituitary peptides. Because of reports of an association between lung maturation and prolactin, 2 different preparations of ovine prolactin were studied in concentrations ranging from 0.1 to 10.0 microgram/ml. Increased saturated phosphatidylcholine (SPC) synthesis without an increase in synthesis of total PC, and increased SPC/PC, was observed after exposure to Sigma prolactin. NIH prolactin in the same concentrations had no effect on synthesis of either PC or SPC. Adrenocorticotropic hormone, luteinizing hormone and thyroid stimulating hormone increased synthesis of both PC and SPC, but none increased the SPC/PC ratio. A cold ethanol extraction of Sigma prolactin yielded fractions with no demonstrable biologic activity, and other initial attempts to characterize the active factor in the Sigma prolactin samples were unsuccessful. We concluded that prolactin per se has no direct effect on PC or SPC synthesis in the lung, but that certain other pituitary peptides have a direct effect on lung synthesis of PC and SPC.