Phospholipase B[EC 3.1.1.5] of Penicillium notatum was shown to catalyze the following two reactions successively: diacylphospholipidlead to 1-monoacylphospholipid+fatty acid; and 1-monoacylphospholipid lead to glycerylphosphoryl-base+fatty acid. 1-Monoacylphospholipid was accumulated measurable in a reaction mixture containing methanol. An attempt to classify phospholipase B according to positional specificity is described.