The origin of oligodendrocytes and astrocytes in the CNS is still a focus of much experimentation and controversy. We have used antibodies against ganglioside GD3 and galactocerebroside (GC) to follow the origin and development of rat cerebellar oligodendrocytes both in vitro and in vivo. The immunofluorescent identification of GC+ cells in the rat neonatal cerebellum in vivo, revealed that cells initially GD3+/GC- appeared to make the transition via GD3+/GC+ cells to GD3-/GC+ oligodendrocytes. This sequence of events closely paralleled the maturation of cerebellar oligodendrocyte precursors found in serum-free dissociated culture. In contrast, whereas both GD3+ and glial fibrillary acidic protein-positive cells were seen in serum-containing dissociated culture and also in freshly dissociated suspensions of cerebellum at postnatal days 0 to 6, such cells could not be identified in situ. Putative GD3+/GC- oligodendrocyte precursor cells arose from the deeper regions of the cerebellum at birth, perhaps initially from the superior medullary velum adjacent to the fourth ventricle, and appeared to migrate into the developing folia just prior to myelination and the acquisition of GC.