Electron microscopy and 3H-thymidine autoradiographic techniques were used to study the fine structure of proliferating cells in developing rat optic nerve. Before the closure of the optic canal almost all of the cells incorporating radioactive thymidine are ventricular cells, but after closure (16 days of gestation) the vast majority are differentiating astroblasts or oligodendroblasts. Labeled astroblasts show a range in their degree of differentiation; some cells lack 90 A cytoplasmic filaments while others have glial filaments and abundant cytoplasmic organelles. In contrast to astroblasts, all of the labeled oligodendroblasts are in the early stages of differentiation. The proliferation of oligodendroblasts starts at five days postnatal, approximately a day or two before the onset of myelination.During myelinogenesis a few of the labeled oligodendroblasts show presumptive connections to myelin sheaths. Microglial cells do not appear to play a major role in gliogenesis since they form less than 2% of all the labeled cells. The results of this study indicate that astroblasts and oligodendroblasts, rather than undifferentiated glioblasts, are the major source of macroglia. The finding that proliferating glia are in the processof differentiation agrees with recent studies which show that differentiated cells can divide.