An on-line thermospray liquid chromatography/mass spectrometry method was developed to rapidly analyze phosphoglyceride molecular species in biological fluid. After total lipid extraction, the extract was subjected to the analysis using on-line reversed-phase high-performance liquid chromatography (HPLC) and filament-on thermospray mass spectrometry. Using nonconventional HPLC conditions, partial separation of individual phospholipid class (phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine) and partial separation of molecular species within each class were achieved. In addition, good peak shape was maintained throughout the analysis. By monitoring characteristic fragment ions (diacylglycerol ions) formed in the filament-on thermospray process and according to the retention time, individual molecular species in each phospholipid class can be identified. Using this method, we observed significant differences in the molecular species composition of each phospholipid class in rat whole-brain lipid. Although nonlinear calibration curves were observed for all the diacylglycerol ions monitored, even in the presence of internal standard, semiquantitative and quantitative results still could be obtained for a mixture of phospholipids.