Astrocyte maturation in the developing corpus callosum and dorsal columns of the spinal cord was studied immunocytochemically in the rat, using antiserum to glial fibrillary acidic protein (GFAP) with a view to determining the relationships of astrocytes to the advancing axons of the corpus callosum and corticospinal tract. Between the eighteenth and nineteenth days of gestation, when the corpus callosum commences forming, most of the GFAP staining in the cerebral hemispheres is contained in radial processes, but some staining of glial cell bodies is also seen in the ventricular zone. At the region of interhemispheric fusion, where the corpus callosum will form, an accumulation of astrocytic processes demonstrable electron microscopically shows light immunocytochemical staining for GFAP. These processes do not adopt a stereotyped orientation. Rather, the overall impression as one moves towards the midline, is of radially disposed processes being disrupted and disoriented by the growing callosal axons at the fusion of the hemispheres. At no time can any orderly arrangement of GFAP-containing processes be seen which might indicate that the processes are serving to guide the growing axons across the midline. There is no immunoreactive staining of cell bodies or processes ventral to the corpus callosum, except in postnatal animals. Prior to the arrival of corticospinal axons in the spinal cord on the first postnatal day (PO)21, GFAP immunoreactivity is greatest in radial processes of the lateral funiculi and in the dorsal median septum. Oblique or vertical processes increase in the cuneate fasciculus from P0 tot P4 but do not appear in the gracile fasciculus until P4. Virtually no stained processes appear in the region to be traversed by the principal corticospinal tract, nor later in the tract itself until late in postnatal development. Only by 3 weeks postnatal is the adult pattern of GFAP staining observed in the corticospinal tract. These results also indicate that the expression of GFAP immunoreactivity is a relatively late phenomenon in astrocytes associated with advancing axons and implies that this aspect of astrocytic maturation is unrelated to any guidance that the immature astrocytes might provide for the growing axons.