Cell line A 549, a continuously cultured line derived from a human pulmonary adenocarcinoma that has morphologic and biochemical features of the pulmonary alveolar type II cell, was studied with regard to saturated phosphatidylcholine synthesis and secretion. Evaluation of a number of culture media showed that those media that supported the most rapid rate of in vitro growth were least optimal for saturated phosphatidylcholine synthesis. Similarly, a labeled precursor (choline labeled with 3H) was most efficiently converted to saturated phosphatidylcholine when growth was slow or arrested but during phases of active cellular proliferation, a greater proportion was incorporated into unsaturated (structural) phosphatidylcholine. De novo synthesis of phosphatidylcholine was primarily via the CDP choline pathway, because no incorporation of labeled methyl groups from S-adenosyl-L-methionine was observed. Cortisol stimulated 3H-labeled choline incorporation into saturated phosphatidylcholine in a manner that likely involved a steroid receptor system but cortisone was inactive. The half-life of cellular saturated phosphatidylcholine was of the order of 100 h and could be accounted for virtually entirely by release of this phospholipid into the medium unchanged. Both cholinergic and adrenergic agents in high doses modestly stimulated the release of pre-labeled saturated phosphatidylcholine from the cells, and the response to these secretagogues was significantly enhanced by cortisol. Cell line A 549 promises to be a useful model system for studies of the cellular events involved in pulmonary saturated phosphatidylcholine synthesis and secretion.