Cell proliferation during successive stages of oligodendrocyte development was delineated in the rat brain and optic nerve. Surface antigens, A2B5, O4, and galactocerebroside (GalC) identified three cell populations emerging in sequence; the incorporation of bromodeoxyuridine into newly synthesized DNA identified the proliferative cells. In vivo, progenitor cells with phenotypes A2B5+O4- and A2B5+O4+GalC- were both proliferative, whereas differentiated GalC+ oligodendrocytes were not. Under basal conditions of culture, the proliferation of both progenitor cell types of the optic nerve was nearly abolished. Activity was restored for A2B5+O4- precursor cells with medium conditioned by either type-1 astrocytes, meningeal cells, or cerebellar interneurons. In contrast, intermediate O4+GalC- cells (proligodendrocytes) were refractory to the astroglial and meningeal signals, but remained as responsive as their precursor cells to the neuronal stimulus. These data further characterize the O4+GalC- proligodendrocyte as a distinct developmental stage, one that specifies a changing response of the cell to environmental mitogens.