AbstractThe unlabeled peroxidase‐anti‐peroxidase method was used to stain glial fibrillary acidic (GFA) protein in the optic nerve of the developing albino rat. Optic nerves from animals ranging in age from the day of birth to adulthood were embedded in paraffin following fixation with various agents for times ranging from 30 minutes to 48 hours. GFA protein activity was demonstrable at birth in large astrocytic processes following fixation with alcohols or with Perfix for short intervals, but not with 4% or 2% buffered paraformaldehyde solutions. With increasing age, GFA protein could be demonstrated using higher aldehyde concentrations, longer fixation times, and longer paraffin embedding schedules. At all ages GFA protein activity was greater following treatment with nonaldehyde fixatives rather than those containing formaldehyde or glutaraldehyde. At birth the majority of GFA protein‐containing processes were confined to planes which were perpendicular to the axons of the optic nerve. With increasing age, tangential and longitudinal processes became more numerous until, in the mature optic nerve, astrocytic processes were best characterized as being multidirectional.