Antiserum against the phosphatidylinositol hapten was obtained in rabbits by injecting a complex of the hapten and methylated bovine serum albumin. The specificity of the antiserum was concerned with the inositol moiety of phosphatidylinositol and was influenced by the ratio of phosphatidylinositol to lecithin. The role of lecithin as an auxiliary lipid in the phosphatidylinositol hapten as well as the cardiolipin hapten was partly explained from observations of the nature of the phospholipase C-treated phospholipid haptens. The lecithin molecule as a whole is indispensable in forming micellar structure of the hapten. However, once the micelle is prepared, the phosphorylcholine moiety of lecithin is no longer necessary, at least in the case of the phosphatidylinositol hapten, and the diglyceride portion of lecithin is important in preserving the micelle already formed. Chemical analysis, reactivity with antisera and binding ability with methylated bovine serum albumin of the enzymetreated phospholipid hapten in comparison with the original hapten supported the above conclusions.