Oligodendrocytes are central nervous system glial cells responsible for myelination of neuronal axons. During brain development oligodendrocyte progenitor cells progress through a series of morphologically and immunohistochemically distinct differentiation steps leading to mature myelin-producing oligodendrocytes. Much of this same differentiation sequence is expressed in vitro by primary oligodendrocyte progenitor cells, and by the clonal progenitor cell line CG-4. We report the use of highly specific monoclonal antibodies against GM1, GD1a, GD1b, GT1b, and GQ1b to determine major brain ganglioside expression and morphological distribution during CG-4 differentiation in vitro. Prominent anti-GD1b antibody stained defined a highly arborized intermediate stage of oligodendrocyte differentiation. In contrast, anti-GT1b antibody bound to discrete patches on the cell bodies of early progenitor cells and more mature oligodendrocytes, and to sites of progenitor arborization. The other anti-ganglioside antibodies tested did not bind above background levels. Cells with anti-GD1b antibody binding and morphology similar to those in differentiating CG-4 cells were detected in rat brain primary cell cultures enriched in oligodendrocyte precursors. The remarkably distinctive ganglioside immunoreactivity on differentiating oligodendrocytes suggests the possibility of a functional role for their surface expression.