A method has been developed for the detection of phospholipase A2 activity following electrophoresis in polyacrylamide gels. A suspension of lecithin trapped in the gel matrix is used as the substrate. Enzyme activity is detected by using rhodamine 6G to stain for unsaturated free fatty acids released by enzyme action (a colorimetric assay) and/or to promote clearing of the lecithin suspension (turbidimetric assay). The procedure is readily capable of detecting 0.01 unit of enzyme activity. No staining is observed in the absence of free calcium ions. The method has been used to determine the composition of electrophoretic variants in phospholipase A2 preparations from mammalian, reptilian, and insect sources. Because different patterns of phospholipase A2 electrophoretic variants were observed in the venoms of snakes of different families, the method also has potential use in blochemical taxonomy.