An improved, rapid, and sensitive method for the biochemical diagnosis of GM1 gangliosidosis based on the detection and quantification of urinary galactosyl-oligosaccharides with high performance liquid chromatography was developed. The oligosaccharides, in 50-100 microliters of urine, were converted to radioactively labeled oligosaccharide-alditols with NaB3H4 and fractionated on commercial silica-amine bonded, high performance liquid chromatography columns. Delineation between infantile, juvenile, and adult onset subtypes of GM1 gangliosidosis was possible by analysis of the levels of the excreted oligosaccharides and their characteristic elution profile. Infantile and juvenile patients contain identical numbers of oligosaccharide fractions (13 resolved components) but can be distinguished by 3-10-fold lower levels of oligosaccharides in juvenile patients and, in some cases by a disproportionately lower concentration of high molecular weight compounds. Adult onset patients were distinguished by substantially lower concentrations of urinary oligosaccharides, 130-180-fold below those in infantile patients, and the apparent absence of high molecular weight oligosaccharides.