Glycosphingolipids in cerebrospinal fluid (CSF) of individual patients with multiple sclerosis (MS) were analyzed using a glycolipid-overlay technique. The ganglioside composition of CSF of non-MS patients was characterized by an abundance of polysialo species, including GT1b and GQ1b. This pattern is completely different from that of human white or gray matter, in which mono- and disialogangliosides predominate. Increased levels of GM1, either associated with or without increases of other gangliosides, such as GD1a, were observed in 16% of the patients with MS (6 of 37 cases: 1 of 15 progressive progressive stage, 4 of 16 progressive stationary stage, and 1 of 6 relapsing stage). The concentration of GD3 was increased in 23% (3 of 13 cases), whereas 1 of 13 cases (8%) showed a dramatic increase of sulfoglucuronyl paragloboside (SGPG) associated with a high level of GD3. These changes may reflect the cellular changes associated with the known pathological lesions in MS, which are characterized by demyelination, gliosis, and/or remyelination with oligodendrocytic proliferation.