A reversed-phase high-performance liquid chromatographic method is described for the separation of individual classes of phospholipids into subfractions. The separation was achieved with isocratic elution using a Nucleosil-5 C18 column and a mobile phase consisting of methanol, acetonitrile and water. Three commercial C18 packing materials were evaluated before Nucleosil was chosen as the stationary phase. The compounds were detected with a combination of an UV detector and a light-scattering mass detector, which provided quantitative chromatograms reported for the first time. The mass detector also allowed location of the most relevant peaks of the UV chromatogram. Phosphatidylcholine and phosphatidylethanolamine species from egg, and phosphatidylcholine species from rat liver, were resolved into more than 20 peaks. The method can be applied to the separation of phospholipids from different biological sources.