The detailed three-dimensional morphology of macroglial cells was determined throughout postnatal development in the intact rat optic nerve, a central nervous system white matter tract. Over 750 cells were analyzed by intracellular injection of horseradish peroxidase or Lucifer Yellow to provide a new perspective of glial differentiation in situ. Retrograde analysis of changes in glial morphology allowed us to identify developmental timetables for three morphological subclasses of astrocytes and oligodendrocytes, and to estimate their time of emergence from undifferentiated glial progenitors. Glial progenitors were recognised throughout postnatal development and persisted in 35-day-old nerves, where we suggest they represent adult progenitor cells. Astrocytes were present at birth, but the majority of these cells developed over the first week as three morphological classes emerged having either transverse, random, or longitudinal process orientation. Several lines of evidence led us to believe that the majority of astrocytes in the rat optic nerve were morphological variations of a single cell type. Young oligodendrocytes were first observed 2 days after birth, indicating that they diverged from progenitors at or near this time. During early development these cells extended a large number of fine processes, which then bifurcated and extended along axons. Later, as myelination proceeded, oligodendrocytes exhibited fewer processes which grew symmetrically and uniformly along the axons, resulting in a highly stereotypic mature oligodendrocyte form. Our analysis of oligodendrocyte growth suggests that these cells did not myelinate axons in a random manner and that axons may influence the myelinating processes of nearby oligodendrocytes. © 1993 Wiley-Liss, Inc.