In freshly dissociated viable cells of early postnatal mouse cerebellum, O1 antigen-positive oligodendrocytes have been eliminated by complement dependent immunocytolysis. Before seeding residual cells in culture, O4 antigen-positive cells were immunolabeled by O4 antibody which had been directly conjugated with rhodamine. After various periods of time in culture, cells were treated with fluorescein conjugated O1 antibody, in order to assay for the appearance of O1-positive oligodendrocytes. After 6.5 h in vitro, the first cells carrying both the previously applied rhodamine label and the freshly applied fluorescein label were seen. The simultaneous appearance of both labels indicates tha O4-positive and O1-negative cells are direct precursors of O1 antigen-positive oligodendrocytes.