A new method for the synthesis of mixed-chain phosphatidylcholines is reported. Silver ion catalyzed acylation of lysophosphatidylcholines by 2-thiopyridyl esters occurs rapidly (10 min) at room temperature in organic solvents. Yields of isomerically pure mixed-chain phosphatidylcholines (>98% isomeric purity) are generally greater than 80%. The reaction proceeds with only 1.5- to 2-fold excess of thiopyridyl ester, thus offering some advantages over existing procedures when precious acylating agents are used. The major disadvantage of the procedure is its sensitivity to water. Phosphatidylcholines having hydroxy fatty acyl groups are prepared by protection of the hydroxyl as the levulinate ester, conversion to the 2-thiopyridyl ester, acylation, and removal of the levulinate with hydrazine. For purification of lysophosphatidylcholines, a reverse-phase high pressure liquid chromatographic method for separation of 1-acylglycerophosphocholines from 2-acylglycerophosphocholines was developed.