Axonal and glial cell development within rat optic nerve in which gliogenesis was altered by systemic injection of 5-azacytidine (5-AZ) was examined by quantitative electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers are premyelinated, and they exhibit a fairly uniform diameter (approximately 0.22 micron). These fibers occupy approximately 55% of the optic nerve volume. At this early age, glia within the optic nerve consist only of cells of astrocytic lineage and progenitor cells. These glia occupy approximately 28% of the optic nerve volume, and there are approximately 80 glial cells/optic nerve cross section. In 14-day-old normal optic nerves, myelinated and ensheathed fibers comprise approximately 17% and 9%, respectively, of the total number of axons. Mean axonal diameter of myelinated fibers is approximately 0.75 micron, while mean diameter for ensheathed axons is approximately 0.50 micron. By volume, these fibers occupy approximately 25% of the nerve, which is similar to the volume occupied by premyelinated axons in these nerves. At 14 days of age, there are approximately 300 glial cells/optic nerve transverse section, and these glia occupy approximately 37% of the volume in normal optic nerve. Oligodendroglia represent approximately 40% of total glial cells present, while astroglia and progenitor cell each comprise approximately 30% of the cells. In optic nerves from 14-day-old rats treated with 5-AZ, few myelinated fibers are present and the number of oligodendroglia is markedly reduced. Axonal diameter of premyelinated fibers is similar to that of age-matched controls. Myelinated and ensheathed fibers comprise approximately 2% of the total fibers present in 5-AZ-treated optic nerves, with the remaining fibers being premyelinated. The few myelinated and ensheathed fibers present in 5-AZ-treated optic nerves display similar axonal diameters to corresponding fibers from age-matched control tissue. Glial cells occupy approximately 40% of the nerve volume, and there are approximately 200 glia/nerve cross section in 5-AZ-treated rats. Astroglia comprise approximately 63% of the total glial cells, while approximately 12% of the cells are oligodendroglia. These results demonstrate that 5-AZ is a potent inhibitor of oligodendrogliogenesis, with a concomitant marked reduction in the number of myelinated fibers.