A new analytical and semi-preparative high-performance liquid chromatographic method for the separation of a brain ganglioside mixture into individual components is described. Gangliosides were applied to a LiChrosorb-NH2 column and eluted with the solvent system acetonitrile-phosphate buffer at different volume ratios and ionic strengths. The elution profile was monitored by flow-through detection of UV absorbance at 215 nm. The separation of mono- to polysialogangliosides was performed in one step in a total elution time lower than 90 min and with high reproducibility.