An improved microscale method for determining lipid phosphorus was developed. Without the prior removal of nonlipid contaminants from crude lipid extracts, the absolute amount of lipid phosphorus and distribution of phospholipids were directly determined by a two-dimensional thin-layer chromatography-internal standard system, using pyrophosphatidic acid as the internal standard. Application of this method to the assay of lipid phosphorus present in small amounts in brain tissues is discussed.