Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material. We studied the synthesis of these two phospholipid classes by alveolar type II cells in primary culture. During a 20-h incubation, type II cells incorporated a high percentage of glycerol, acetate, and palmitate into phosphatidylcholine (61.2, 76.4, and 76.8% of lipid radioactivity, respectively) and into phosphatidylglycerol (16.7, 5.8, and 6.6%). Acetate was incorporated principally by de novo synthesis of fatty acids rather than by chain elongation. We studied the pathways for synthesis of saturated phosphatidylcholine and phosphatidylglycerol with type II cells that had been in culture for 1 day. Palmitate was incorporated nearly equally into positions 1 and 2 of saturated phosphatidylglycerol, but predominantly (72%) into position 2 of saturated phosphatidylcholine. These data imply that saturated phosphatidylcholine is synthesized at least in part by acylation of 1-acyl-2-lysophosphatidylcholine. Alveolar type II cells also incorporated a mixture of saturated 1-[9,10-3H]palmitoyl-2-lysophosphatidylcholine and 1-acyl-2-lysophosphatidyl-[1,2-14C]choline from the medium by direct acylation rather than by transacylation. As the duration of culture increased beyond 1 day, type II cells incorporated a lower percentage of palmitate into phosphatidylglycerol and saturated phosphatidylcholine.