A procedure for the phospholipase A2 hydrolysis of phosphatidylcholine on a thin-layer chromatographic plate and subsequent separation of the products on the same plate is described. A 0.2-0.8-mg amount of Russell's viper venom (phospholipase A2) in 0.2 ml of 0.005 M calcium chloride solution was applied on a 0.5-mm silica gel G plate as a band over which 2-5 mg of egg phosphatidylcholine in 0.2 ml of diethyl ether containing 5% of methanol was evenly applied. After the reaction had proceeded for 15-20 min in a diethyl ether-saturated chamber at 25°, the plate was developed with chloroform-methanol-water (65:25:4). The bands were identified and their contents extracted. The extent of hydrolysis under different reaction conditions was evaluated from the amount of lysophosphatidylcholine formed. Approximately 74.6% (maximum) conversion was obtained within 15 min at 25° using substrate to enzyme ratio of 4:1. The acyl group distribution in the 1- and 2-positions of hen egg phosphatidylcholine obtained from the gasliquid chromatographic analysis of the methyl ester corresponding to the lyso and free fatty acid band agreed with those obtained by the method of Wells and Hanahan. The method is also applicable to phosphatidylethanolamine. © 1979.