An improved method for the partial chemical synthesis of unsaturated and radioactively labeled phosphatidylcholines is described. This procedure offers advantages over conventional acylation methods in that it can be carried out on a millimole or micromole scale under mild conditions and it does not require a large excess of the fatty acid acylating reagent. In this procedure sn-glycero-3-phosphorylcholine is reacted with twice the theoretical amount of fatty acid imidazolide and sodium methylsulfinylmethide in dimethylsulfoxide for several minutes at 17 degrees C. Phosphatidylcholine, which was purified by gradient-elution chromatography on silicic acid, was isolated in 60% yield and was estimated to be about 99% pure. The preparations of 1,2-dioleoyl-, 1,2-dilinoleoyl-, and 1,2-dilinolenoyl-sn-glycero-3-phosphorylcholine are described. The reaction was also carried out on a small scale for the preparation of high specific activity 1,2-di[ 1'(-14)C]oleoyl-sn-glycero-3-phosphorylcholine in 38% yield with a specific activity of about 9.7 muCi/mumol.