The phenotypic expression of gangliosides and galactolipids was investigated using primary cultures of fetal human oligodendrocytes and astrocytes. These glial cells were isolated from fetal human brains of 12-18 weeks' gestation. Expression of gangliosides and galactolipids in oligodendrocytes and astrocytes was investigated by double labeling immunocytochemistry using rabbit antibodies specific for galactocerebroside (GalC, a cell type-specific marker for oligodendrocyte) and glial fibrillary acidic protein (GFAP, a cell type-specific marker for astrocyte) in combination with a panel of mouse monoclonal antibodies which react with specific gangliosides or galactolipids. A considerable number of GalC+ oligodendrocytes expressed intense immunoreactivities specific for GM3 (19%) and GM2 (45%) gangliosides. Approximately 11% of GalC+ oligodendrocytes expressed GM4 immunoreactivity, and smaller numbers of GalC+ oligodendrocytes expressed GD3 (4%), GD2 (1%), GT1b (5%) and A2B5 (3%) immunoreactivities. However, GalC+ oligodendrocytes did not express GM1, GD1a, GT1b or GQ1c. Major populations of GalC+ oligodendrocytes immunolabeled by rabbit anti-GalC antibody reacted with anti-GalC mAb (Ranscht mAb, 81%) or by anti-sulfatide mAb (O4 mAb, 91%). A considerable number of GFAP+ astrocytes expressed intense GM2 (26%) and GD2 (15%) immunoreactivities, while a smaller population expressed intense GM3 (3%), GD3 (6%) and GM4 (4%) immunoreactivities. Weak immunoreactions specific for GD1b, A2B5 and sulfatide were found in less than 1% each of GFAP+ astrocytes, while GFAP+ astrocytes did not express GM1, GD1a, GT1a, GT1b or GQ1b. These results indicate that GM3, GM2 and sulfatide are expressed in a major population of GalC+ oligodendrocytes, while GM3, GM2, GD3, GD2, and GM4 are expressed in a small but distinctive population of GFAP+ astrocytes. Our results suggest that GM4, GM1 and GD3, which are utilized as markers for adult human oligodendrocytes and myelin, are not the major ganglioside constituents in cultured fetal human oligodendrocytes.