The kinetics of labeling of lung phosphatidylcholine and disaturated phosphatidylcholine were studied for periods from 0.75--120 min following intravenous injection of radiolabeled palmitic acid and choline into 3-day-old rabbits. The labeled palmitic acid was cleared rapidly from plasma, and rapidly appeared with identical incorporation kinetics in both phosphatidylcholine and disaturated phosphatidylcholine. The 2-acyl positions of both phosphatidylcholine and disaturated phosphatidylcholine were labeled preferentially soon after [14C]palmitic acid injection. The specific activities of palmitic acid in the 2-acyl positions of phosphatidylcholine and disaturated phosphatidylcholine 0.75 min after injection of labeled palmitic acid were 3.4 and 1.9 times, respectively, the specific activities of palmitic acid in the 1-acyl positions. By 120 min the label had randomized between the 1-acyl and 2-acyl positions, and the kinetics of that randomization were defined for both phosphatidylcholine and disaturated phosphatidylcholine. Choline did not pulse label lung phosphatidylcholine or disaturated phosphatidylcholine. The choline label appeared with equal specific activities in both phosphatidylcholine and disaturated phosphatidylcholine. Thus no analysis of the de novo synthesized product via the CDP-choline pathway was possible.