Automated multiple development chromatography has been utilized to enhance separation of gangliosides on silica-gel precoated high-performance TLC plates. Three-fold chromatography of a complex mixture of neolacto-series monosialogangliosides in the solvent chloroform-methanol-water (120:85:14, v/v, 2 mM CaCl2) resulted in a ca. three-fold increase in separation distance of e.g. alpha 2-3 and alpha 2-6 sialylated ganglioside isomers compared to conventional single chromatography in the standard solvent chloroform-methanol-water (120:85:20, v/v, 2 mM CaCl2). An extremely heterogenous murine disialoganglioside mixture was developed three times in chloroform-methanol-water (120:85:16, v/v, 2 mM CaCl2) leading to a more than two-fold increase in separation distance. Chloroform-methanol-water (120:85:22, v/v, 2 mM CaCl2) was the solvent of choice for multiple chromatography of ganglio-series polysialogangliosides from embryonic chicken brain.