Abstract The effects of phospholipase A 2 and phospholipase C on Bacillus subtilis protoplast membrane have been studied by electron microscopy and by chemical methods. Phospholipase A 2 (from porcine pancreas) almost quantitatively converted cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol to fatty acids and lysoderivatives. The fatty acids like the lysophospholipids remained in the membrane. Phospholipase C (from Bacillus cereus) hydrolyzed about 80% of the phosphatidylethanolamine and about 40% of the cardiolipin. Electron microscopy has been carried out with respect to general morphology of the affected protoplasts, the occurrence of a triple-layered membrane structure in thin sections, and the ultrastructure of membrane fracture faces upon freeze fracturing. Phospholipase A 2 treatment resulted in fragmentation of the protoplasts. In all cases the triple-layered membrane profile was preserved in thin sections. The membrane fracture faces appeared normal, i.e. they showed a convex face with many particles and a concave face with few particles. This indicated that the hydrophobic interior of the membrane was not too much damaged after incubation with phospholipases, presumably because of the stabilizing action of membrane proteins.