Synthesis of lecithins in isolated type II alveolar cells was compared with that in alveolar macrophages as a means of exploring the biochemical mechanisms underlying surfactant production in the lung. Counted cell populations were suspended in a simple glucose-salt solution and 14C-labeled precursors were added singly, in physiologic concentrations, to assess the potential importance of each as a substrate for lecithin synthesis. Molar incorporation of glucose, glycerol, choline, lysolecithin, acetate, palmitate, oleate, and linoleate was determined in lecithins fractionated according to degree of saturation after 1 hour of incubation. Palmitate ws the most actively utilized substrate in type II &cells. Type II cells incorporated 6 nmoles of palmitate per 10(7) cells, of which 77% was in disaturated lecithins, and 66% at the C2 position (compared to 0.8 nmoles, 47% disaturated, in macrophages). Acetate was also incorporated mainly into disaturated lecithins in type II cells; macrophages did not utilize acetate, and no precursor specifically supported disaturated lecithin synthesis in macrophages. Type II cells and macrophages synthesized similar quantities of total lecithins and disaturated lecithins from glucose and choline. Only the type II cells, however, were capable of increasing disaturated lecithin synthesis from 14C-choline when unlabeled palmitate was added to the medium. Type II cells synthesized significantly more disaturated lecithins from lysolecithin than did macrophages (451 versus 60 pmoles per 10(7) cells). Macrophages utilized glycerol in lecithin synthesis, but type II cells did not. Our data demonstrate directly for the first time that type II cells are the site of disaturated lecithin synthesis and that acyl turnover mechanisms are important in production of disaturated lecithins by the type II cell.